Water-borne outbreak of oropharyngeal and glandular tularemia in Georgia: investigation and follow-up.

BACKGROUND: In November 2006, an outbreak of waterborne tularemia occurred in an eastern region in the Republic of Georgia. Outbreak investigation revealed 26 cases: 21 oropharyngeal and 5 glandular tularemia cases. METHODS: The presentation of the index case triggered an outbreak investigation involving the collection of clinical/ epidemiological data, application of tularine skin test, and laboratory confirmation of the possible cases using the tube agglutination test and polymerase chain reaction (PCR) testing. Serology results were verified by enzyme-linked immunosorbent assay (ELISA) and Western blot. A case- control study along with follow-up was conducted 4 months after the index case presentation. RESULTS: Exudative pharyngitis, predominantly laterocervical adenitis, fever, and headache were the most prevalent clinical signs/symptoms observed. Depressed mood, concentration difficulties, and sleep disturbance were also detected. Bubo aspirates tested by PCR were positive in 4/4 cases and pharyngeal swabs also tested by PCR were positive in 2/3 cases. Francisella tularensis was isolated from the water samples. Comparison of the cases and controls did not reveal any statistically significant risk factors. A follow-up investigation revealed cases with protracted symptoms of fatigue, headache, and sleep disturbance. Additionally, 8/26 cases still had cervical adenopathy of prominent size. A delay in diagnosis was associated with persistent lymphadenopathy on follow-up examination (p = 0.05). CONCLUSION: We observed unique features of persistent neuropsychiatric symptoms and lymphadenopathy 5 months after tularemia infection which were associated with delayed diagnosis and the lack of prompt response to therapy. This outbreak of oropharyngeal tularemia emphasizes the importance of a rapid diagnostic and investigative response to tularemia. This type of response can prevent ongoing exposure, as well as provide expeditious treatment to mitigate persistent symptoms.

The Drosophila melanogaster ade5 gene encodes a bifunctional enzyme for two steps in the de novo purine synthesis pathway.

Steps 6 and 7 of de novo purine synthesis are performed by 5-aminoimidazole ribonucleotide carboxylase (AIRc) and 4-[(N-succinylamino)carbonyl]-5-aminoimidazole ribonucleotide synthetase (SAICARs), respectively. In vertebrates, a single gene encodes AIRc-SAICARs with domains homologous to Escherichia coli PurE and PurC. We have isolated an AIRc-SAICARs cDNA from Drosophila melanogaster via functional complementation with an E. coli purC purine auxotroph. This cDNA encodes AIRc yet is unable to complement an E. coli purE mutant, suggesting functional differences between Drosophila and E. coli AIRc. In vertebrates, the AIRc-SAICARs gene shares a promoter region with the gene encoding phosphoribosylamidotransferase, which performs the first step in de novo purine synthesis. In Drosophila, the AIRc-SAICARs gene maps to section 11B4-14 of the X chromosome, while the phosphoribosylamidotransferase gene (Prat) maps to chromosome 3; thus, the close linkage of these two genes is not conserved in flies. Three EMS-induced X-linked adenine auxotrophic mutations, ade4(1), ade5(1), and ade5(2), were isolated. Two gamma-radiation-induced (ade5(3) and ade5(4)) and three hybrid dysgenesis-induced (ade5(5), ade5(6), and ade5(8)) alleles were also isolated. Characterization of the auxotrophy and the finding that the hybrid dysgenesis-induced mutations all harbor P transposon sequences within the AIRc-SAICARs gene show that ade5 encodes AIRc-SAICARs.

The purine biosynthesis enzyme PRAT detected in proenzyme and mature forms during development of Drosophila melanogaster.

Glutamine phosphoribosylpyrophosphate amidotransferase (PRAT; EC 2.4. 2.14) is the first and rate-limiting enzyme of de novo purine biosynthesis. PRAT expression in Drosophila development was examined to determine if it is correlated with cell proliferation and/or nutritional isolation. An antiserum, raised against the 16 carboxyl-terminal amino acids of PRAT, detects two proteins corresponding to a 60 kDa proenzyme and 55 kDa mature enzyme, consistent with a 53 amino acid propeptide predicted from the gene sequence. Mature enzyme is maternally expressed, and proenzyme appears in embryos at 2-8 h, corresponding to the interval during which zygotic transcription is initiated. Upon hatching of first instar larvae, proenzyme levels are reduced and remain low relative to mature enzyme. Adult females have higher levels of both proteins relative to males, consistent with maternal expression. Maternal expression reflects a requirement for the enzyme during embryogenesis, while reduction in expression following hatching reflects a switch to an exogenous source of purines. Prat mRNA levels follow a similar overall pattern in the same developmental stages examined for the protein. Discovery of a second gene encoding PRAT with 78% amino acid identity leads to the possibility that the antiserum raised against the carboxyl-terminus detects two enzymes.

Repetitive arrays containing a housekeeping gene have altered polytene chromosome morphology in Drosophila.

Much of our understanding of gene and chromatin organization has been developed from observation of polytene chromosomes. We describe an experimental approach using transgenes that has allowed us to observe local changes in polytene morphology. A composite P transposon that contains a fusion between the regulatory region of Prat, a purine synthesis gene, and brown (bw), an eye pigment reporter, was transformed into the 65A10 polytene band and subjected to P-transposase mutagenesis. Arrays of up to 320 kb at 65A10 were recovered by selection for increased pigment, and pigment levels were found to be proportional to numbers of copies. In polytene chromosomes, the original transformant was found to split 65A10 into two thin bands separated by an interband. With increases in copy number, the interband became progressively denser, eventually forming a dark, amorphous, deformable structure unlike any previously reported. The persistence of Prat expression in development, together with the cytological appearance of these large arrays, suggest that the state of the Prat promoter is affecting polytene structure. Because this unique structure is distinct from bands, interbands, puffs, and the chromocenter, which comprise polytene chromosomes, we suggest that it is composed of an altered form of chromatin.

Introduction of a DNA methyltransferase into Drosophila to probe chromatin structure in vivo.

The dam DNA methyltransferase gene from Escherichia coli was introduced into Drosophila in order to probe chromatin structure in vivo. Expression of the gene caused no visible defects or developmental delay even at high levels of active methylase. About half of each target site was found to be methylated in vivo, apparently reflecting a general property of chromatin packaged in nucleosomes. Although site-specific differences were detected, most euchromatic and heterochromatic sites showed comparable degrees of methylation, at least at high methylase levels. Methylase accessibility of a lacZ reporter gene subject to position-effect variegation throughout development was only slightly reduced, consistent with studies of chromatin accessibility in vitro. Silencing of lacZ during development differed from silencing of an adjacent white eye pigment reporter gene in the adult, as though chromatin structure can undergo dynamic alterations during development.

Molecular cloning and characterization of the dpy-20 gene of Caenorhabditis elegans.

We describe the molecular analysis of the dpy-20 gene in Caenorhabditis elegans. Isolation of genomic sequences was facilitated by the availability of a mutation that resulted from insertion of a Tc1 transposable element into the dpy-20 gene. The Tc1 insertion site in the m474::Tc1 allele was identified and was found to lie within the coding region of dpy-20. Three revertants (two wild-type and one partial revertant) resulted from the excision of this Tc1 element. Genomic dpy-20 clones' were isolated from a library of wild-type DNA and were found to lie just to the left of the unc-22 locus on the physical map, compatible with the position of dpy-20 on the genetic map. Cosmid DNA containing the dpy-20 gene was successfully used to rescue the mutant phenotype of animals homozygous for another dpy-20 allele, e1282ts. Sequence analysis of the putative dpy-20 homologue in Caenorhabditis briggsae was performed to confirm identification of the coding regions of the C. elegans gene and to identify conserved regulatory regions. Sequence analysis of dpy-20 revealed that it was not similar to other genes encoding known cuticle components such as collagen or cuticulin. The dpy-20 gene product, therefore, identifies a previously unknown type of protein that may be directly or indirectly involved in cuticle function. Northern blot analysis showed that dpy-20 is expressed predominantly in the second larval stage and that the mRNA is not at all abundant. Data from temperature shift studies using the temperature-sensitive allele e1282ts showed that the sensitive period also occurs at approximately the second larval stage. Therefore, expression of dpy-20 mRNA and function of the DPY-20 protein are closely linked temporally.

Molecular and genetic analyses of Drosophila Prat, which encodes the first enzyme of de novo purine biosynthesis.

The Drosophila Prat gene encodes phosphoribosylamidotransferase (PRAT), the enzyme that performs the first committed step of the de novo purine nucleotide biosynthesis pathway. Using information from amino acid sequence alignments of PRAT from other organisms, a polymerase chain reaction-based approach was employed to clone Prat. Amino acid sequence alignment of Drosophila PRAT with PRAT from bacteria, yeast, and vertebrates indicates that it is most identical (at least 60%) to the vertebrate PRATs. It shares putative amino-terminal propeptide and iron-binding domains seen only in Bacillus subtilis and vertebrate PRATs. Prat was localized to the right arm of chromosome 3 at polytene band 84E1-2. Owing to the fact that this region had been well characterized previously, Prat was localized to a 30-kilobase region between two deficiency breakpoints. By making the prediction that Prat would have a similar "purine syndrome" phenotype as mutations in the genes ade2 and ade3, which encode enzymes downstream in the pathway, five alleles of Prat were isolated. Three of the alleles were identified as missense mutations. A comparison of PRAT enzyme activity with phenotype in three of the mutants indicates that a reduction to 40% of the wild-type allele's activity is sufficient to cause the purine syndrome, suggesting that PRAT activity is limiting in Drosophila.

Unusual organizational features of the Drosophila Gart locus are not conserved within Diptera.

The Drosophila Gart locus consists of two genes. One gene encodes three enzymes in the de novo purine nucleotide biosynthesis pathway [glycinamide ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide synthetase (AIRS), and glycinamide ribonucleotide transformylase (GART)]. The second gene lies within an intron of the purine gene and encodes a cuticle protein. To investigate the evolution of the Gart locus, the Chironomus tentans homolog was cloned by screening a genomic DNA library with a polymerase chain reaction product. This study shows that the interesting structural features of this locus conserved in two distant Drosophila species are not found in the Chironomus homolog. These features include the cuticle protein gene nested within an intron and the existence of an alternative transcript to yield a monofunctional enzyme. In addition, the extremely rapid divergence of coding sequence seen for members of the tandemly duplicated AIRS domain in Drosophila is found to be much less rapid in Chironomus.

Genetic analysis and complementation by germ-line transformation of lethal mutations in the unc-22 IV region of Caenorhabditis elegans.

The subject of this study is the organization of essential genes in the 2 map-unit unc-22 IV region of the Caenorhabditis elegans genome. With the goal of achieving mutational saturation of essential genes in this region, 6491 chromosomes mutagenized with ethyl methanesulfonate (EMS) were screened for the presence of lethal mutations in the unc-22 region. The genetic analysis of 21 lethal mutations in the unc-22 region resulted in the identification of 6 new essential genes, making a total of 36 characterized to date. A minimum of 49 essential genes are estimated to lie in this region. A set of seven formaldehyde-induced deficiencies of unc-22 and surrounding loci were isolated to facilitate the positioning of essential genes on the genetic and physical maps. In order to study essential genes at the molecular level, our approach was to rescue lethal mutations by the injection of genomic DNA in the form of cosmid clones into the germ-line of balanced heterozygotes carrying a lethal mutation. The cosmid clones containing let-56 and let-653 were identified by this method.

Genetic and fine structure analysis of unc-26(IV) and adjacent regions in Caenorhabditis elegans.

The genetic organization of unc-26(IV) and adjacent regions was studied in Caenorhabditis elegans. We constructed a fine structure genetic map of unc-26(IV), a gene that affects locomotion and pharyngeal muscle movement but not muscle structure. Eleven alleles were positioned relative to each other recombinationally and were classified according to phenotypic severity. The unc-26 gene spans at least 0.026 map units, which is exceptionally large for a C. elegans gene. All but one allele, e205, are amorphic alleles. Interestingly, e205 is hypomorphic but also suppressible by the amber suppressor sup-7. Nineteen lethal mutations in the unc-26 region were isolated and characterized. The unc-26 region is subdivided into four zones by five deficiency breakpoints. These mutations fall into 15 complementation groups. The stages of development affected by these mutations were determined.

Analysis of lethal mutations induced in a mutator strain that activates transposable elements in Caenorhabditis elegans.

A screen was conducted for lethal mutations in the nematode Caenorhabditis elegans in a strain containing the mutator mut-4 (st700)I to examine the nature of mutator-induced lethal mutations within two large chromosomal regions comprising a total of 49 map units (linkage group IV (right) and linkage group V (left)). The genetic analysis of 28 lethal mutations has revealed that the mutator locus mut-4(st700)I causes both putative single-gene mutations and deficiencies. We have identified lethal mutations in three different genes, in addition to seven deficiencies. There is a mutational hot spot on linkage group V (left) around the lin-40 locus. Six mutations appear to be alleles of lin-40. In addition, 5 of 7 deficiencies have breakpoints at or very near lin-40. All seven deficiencies delete the left-most known gene on linkage group V (left) and thus appear to delete the tip of the chromosome. This is in contrast to gamma ray and formaldehyde induced deficiencies, which infrequently delete the closest known gene to the tip of a chromosome.

The unc-22(IV) region of Caenorhabditis elegans: genetic analysis of lethal mutations.

The organization of essential genes in the unc-22 region, defined by the deficiency sDf2 on linkage group IV, has been studied. Using the balancer nT1 (IV;V), which suppresses recombination over 49 map units, 294 lethal mutations on LGIV(right) and LGV(left) were recovered using EMS mutagenesis. Twenty-six of these mutations fell into the unc-22 region. Together with previously isolated lethal mutations, there is now a total of 63 lethal mutations which fall into 31 complementation groups. Mutations were positioned on the map using eight overlapping deficiencies in addition to sDf2. The lethal alleles and deficiencies in the unc-22 region were characterized with respect to their terminal phenotypes. Mapping of these lethal mutations shows that sDf2 deletes a minimum of 1.8 map units and a maximum of 2.5 map units. A minimum estimate of essential gene number for the region using a truncated Poisson calculation is 48. The data indicate a minimum estimate of approximately 3500 essential genes in the Caenorhabditis elegans genome.