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PubMed2004

Genetic characterization of the Guinea-Bissau family of Mycobacterium tuberculosis complex strains.

In a previous study of Mycobacterium tuberculosis complex isolates from Guinea-Bissau in West Africa, we identified a unique group of strains, designated here as the Guinea-Bissau family of strains, which, although genotypically closely related, phenotypically demonstrated a considerable heterogeneity. We conducted here a detailed genotypic analysis of a subset (n = 35) of these isolates. Based on the data obtained, and by comparison of known corresponding genes in mycobacteria outside the M. tuberculosis complex, we propose that the Guinea-Bissau strains belong to a unique branch of the M. tuberculosis complex tree in between classical M. tuberculosis and classical M. bovis. These observations are discussed in their significance in M. tuberculosis complex classification.

Amidohydrolases↗

PubMedDate not supplied

Genetic characterization of wild-type measles viruses isolated during the 1998 measles epidemic in Argentina.

This study reports on genomic characterization of six measles virus (MV) isolates obtained from a measles epidemic in Argentina in 1998. Reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequence analysis of the carboxyl-terminal region of the nucleoprotein (N) gene of these isolates classified all of them as wild type MV of D6 genotype. MVs of D6 genotype with identical nucleotide sequences in the region analyzed were also identified during the 1997 measles epidemic in Brazil and the 1999 measles outbreak in Uruguai. These results suggest that the MVs associated with the 1998 measles epidemic in Argentina might have originated from Brazil. As the D6 genotype is also widely distributed in Europe, it is possible that this genotype was brought to South America from Europe.

Adult↗

PubMed1976

Genetic characterization of methotrexate-resistant chinese hamster ovary cells.

In a previous report, we described the selection and partial characterization of three methotrexate (Mtx)-resistant Chinese hamster ovary cells (CHO) (1). Class I cells contained an apparent structural alteration in dihydrofolate reductase. Class II cells had an alteration affecting the permeability of the drug. Class III cells, selected from Class I cells, had an increased activity of the altered enzyme. In the work described here, it has been shown that the spontaneous mutation rate to Class I resistance is in the order of 2 X 10-9 mutations per locus per generation and that in single-step mutagenized selections the number of resistant colonies of Class I and II are about equal. Class I and Class III resistance is expressed codominantly in somatic cell hybrids, whereas the Class II resistant marker is a recessive trait.

Animals↗

PubMed2002

Genetic characterization and sequence heterogeneity of a canadian isolate of Swine hepatitis E virus.

Swine hepatitis E virus (HEV) is a newly identified potentially zoonotic agent that is possibly transmitted to humans from pigs. Swine HEV is prevalent in pig populations and does not cause abnormal clinical symptoms in infected pigs, further implicating a likelihood of a risk of transmission to humans by normal contact. To date in North America, only one strain of swine HEV (strain US swine) has been fully sequenced. In the present study, we identified a swine HEV isolate from pigs in Canada, designated the Arkell strain, and determined the full length of the genomic sequence. The genome of Canadian strain Arkell consisted of 7,242 nucleotides, excluding the poly(A) tail of at least 15 A residues. The genome contained three open reading frames (ORFs), ORF1, ORF2, and ORF3, which had coding capacities for proteins of 1,708, 660, and 122 amino acids, respectively. Comparative analysis of the full-length genomic sequence indicated that the sequence of strain Arkell was distinct from those of all other known HEV isolates by 13 to 27% and shared the highest degrees of identity with human HEV isolates US-1 and US-2, HEV isolate US swine, and the human and swine HEV isolates recently isolated in Japan. On the basis of sequence similarities and phylogenetic analyses, HEV strain Arkell was grouped into genotype 3. The sequence of the Arkell swine HEV isolate differed from those of HEV isolate US swine and HEV isolate Japan swine by 13 and 14%, respectively. To date, two isolates of swine HEV (isolates Arkell and SK3 [D. Yoo et al., Clin. Diagn. Lab. Immunol. 8:1213-1219, 2001]) have been identified in Canadian pigs, and their sequences also differ from each other by 11.8%. Our studies indicate that, as with human HEV strains, swine HEV isolates exhibit extensive genetic heterogeneity.

3' Untranslated Regions↗

PubMed2006

Genetic characterization of the chlorothricin gene cluster as a model for spirotetronate antibiotic biosynthesis.

The biosynthetic gene cluster for chlorothricin (CHL) was localized to a 122 kb contiguous DNA from Streptomyces antibioticus DSM 40725, and its involvement in CHL biosynthesis was confirmed by gene inactivation and complementation. Bioinformatic analysis of the sequenced 111.989 kb DNA region revealed 42 open reading frames, 35 of which were defined to constitute the CHL gene cluster. An assembly model for CHL biosynthesis from D-olivose, 2-methoxy-5-chloro-6-methylsalicyclic acid, and chlorothricolide building blocks was proposed. This work represents cloning of a gene cluster for spirotetronate antibiotic biosynthesis and sets the stage to investigate the unusual macrolide biosynthesis including tandem Diels-Alder cyclizations, Baeyer-Villiger oxidation, and incorporation of an enoylpyruvate unit.

Amino Acid Sequence↗

PubMed2005

First biologic and genetic characterization of Toxoplasma gondii isolates from chickens from Africa (Democratic Republic of Congo, Mali, Burkina Faso, and Kenya).

The prevalence of Toxoplasma gondii in free-ranging chickens (Gallus domesticus) is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. In the present study, prevalence of T. gondii in chickens from Democratic Republic of Congo, Mali, Burkina Faso, and Kenya is reported. The prevalence of T. gondii antibodies in sera of 50 free-range chickens from Congo was 50% based on the modified agglutination test (MAT); antibody titers were 1:5 in 7, 1:10 in 7, 1:20 in 6, 1:40 in 1, and 1:160 or more in 4 chickens. Hearts, pectoral muscles, and brains of 11 chickens with titers of 1:20 or more were bioassayed individually in mice; T. gondii was isolated from 9, from the hearts of 9, brains of 3, and muscles of 3 chickens. Tissues of each of the 14 chickens with titers of 1:5 or 1:10 were pooled and bioassayed in mice; T. gondii was isolated from 1 chicken with a titer of 1:10. Tissues from the remaining 25 seronegative chickens were pooled and fed to 1 T. gondii-free cat. Feces of the cat were examined for oocysts, but none was seen. The results indicate that T. gondii localizes in the hearts more often than in other tissues of naturally infected chickens. Genotyping of these 10 isolates using the SAG2 locus indicated that 8 were isolates were type III, 1 was type II, and 1 was type I. Two isolates (1 type I and 1 type III) were virulent for mice. Toxoplasma gondii was isolated by mouse bioassay from a pool of brains and hearts of 5 of 48 chickens from Mali and 1 of 40 chickens from Burkina Faso; all 6 isolates were avirulent for mice. Genetically, 4 isolates were type III and 2 were type II. Sera were not available from chickens from Mali and Burkina Faso. Toxoplasma gondii antibodies (MAT 100 or more) were found in 4 of 30 chickens from Kenya, and T. gondii was isolated from the brain of 1 of 4 seropositive chickens; this strain was avirulent for mice and was type II. This is the first report on isolation and genotyping of T. gondii from any source from these 4 countries in Africa.

Agglutination Tests↗

PubMed1988

Genetic characterization of hyperresistance to formaldehyde and 4-nitroquinoline-N-oxide in the yeast Saccharomyces cerevisiae.

The hyperresistance to 4-nitroquinoline-N-oxide (4-NQO) and formaldehyde (FA) of yeast strains transformed with the multi-copy plasmids pAR172 and pAR184, respectively, is due to the two genes, SNQ and SFA, which are present on these plasmids. Restriction analysis revealed the maximal size of SFA as 2.7 kb and of SNQ as 2.2 kb, including transcription control elements. The presence of the smallest 2.7 kb subclone carrying SFA increased hyperresistance to formaldehyde fivefold over that of the original pAR184 isolate. No such increase in hyperresistance to 4-NQO was seen with the smaller subclones of the pAR172 isolate. Disruption of the SFA gene led to a threefold increase in sensitivity to FA as compared with the wild type. Expression of gene SNQ introduced on a multi-copy vector into haploid yeast mutants rad2, rad3, and snm1 did not complement these mutations that block excision repair.

4-Nitroquinoline-1-oxide↗

PubMed1993

Genetic characterization of plasmid-encoded multiple antibiotic resistance in a strain of Listeria monocytogenes causing endocarditis.

One susceptible and two multiply resistant isolates of Listeria monocytogenes from a patient suffering from prosthetic valve endocarditis are described. They could not be distinguished by several typing methods. Two isolates were resistant to chloramphenicol, macrolide/lincosamide/streptogramin antibiotics and tetracycline. The resistance determinants were located on a 39 kb plasmid pWDB100 that was transferable by filter mating to several gram-positive bacteria. Evidence was obtained to support the hypothesis that the resistant variant had primarily infected the patient's blood and prosthetic valve, and later lost the resistance plasmid. The three resistance determinants showed homology to other known markers, cat221/cat223, ermB and tetM, which are frequently found in different gram-positive genera. Plasmid pWDB100 showed extensive homology to the Streptococcus agalactiae broad-host-range plasmid pIP501. It was also very similar to two listerial plasmids found in France. Thus, plasmid pWDB100 and the homologous plasmids from France, although isolated in geographically distant regions, may illustrate spread of a plasmid and its relatives.

Conjugation, Genetic↗

PubMed1998

Nucleotide sequence and genetic characterization of the novel IncQ-like plasmid pIE1107.

The analysis of the complete nucleotide sequence of the small resistance plasmid pIE1107 revealed a close similarity to the well-known IncQ plasmids. Highly conserved replication proteins and nearly identical origins of replication (oriV) suggest equivalent functions in the related replication systems. However, pIE1107 contains two copies of IncQ-oriV-like DNA which are slightly different regarding the iterons. Upon deletion of a silent copy of IncQ-oriV-like DNA the resulting plasmid is fully compatible with IncQ plasmids, indicating that there is no mutual communication between the replication control of the respective replicons. Experiments with cloned oriV DNA strongly suggest that the replication initiation protein of pIE1107 has specialized into the distinct target-iterons of its own oriV which differs only by a few nucleotides from the oriV of IncQ plasmids. Implications from the apparent highly specific protein-DNA recognition and from the incompatibility properties of pIE1107 for the evolution of a family of compatible, IncQ-like plasmids are discussed.

Amino Acid Sequence↗

PubMed1999

Genetic characterization and evolutionary implications of a chromosomally encoded naphthalene-degradation upper pathway from Pseudomonas stutzeri AN10.

Pseudomonas stutzeri strain AN10 is a naphthalene-degrading strain whose dissimilatory genes are chromosomally encoded. We sequenced a total of 11514bp including the entire naphthalene-degradation upper pathway (nah) of P. stutzeri AN10. Nine open reading frames, nahAaAbAcAdBFCED, encoding the enzymes for the degradation of naphthalene to salicylate, were identified. The nah genes of P. stutzeri AN10 have been compared with genes encoding isofunctional proteins from other Pseudomonas naphthalene-degradation upper pathways. The implications of the sequence homologies to the evolution of aromatic catabolic pathways are discussed. Our findings indicate that this entire catabolic module of P. stutzeri AN10 was recruited from other microorganisms and a short period of time has elapsed after its incorporation within the P. stutzeri AN10 genome. Comparisons also suggest the coexistence of two entire nah upper pathways in a host strain, and further recombination between them. These events could accelerate the evolution of modern catabolic pathways.

Amino Acid Sequence↗

PubMed1983

Isolation and genetic characterization of temperature-sensitive mutants that define five additional recombination groups in simian rotavirus SA11.

Nineteen independent temperature-sensitive (ts) mutants were isolated from SA11 following mutagenesis with proflavin or 5-azacytidine. Fourteen of the ts mutants fell into one or another of five mutant groups previously defined by recombination. Five of the ts mutants defined five recombination groups (F, G, H, I, and J) that had not been previously identified. Thus, 10 of the 11 expected mutant groups have been identified in SA11. The prototype mutants of the 10 mutant groups were tested for recombination at nonpermissive temperature to determine if any group had a lesion affecting recombination. Most mutant pairs recombined efficiently; however, the tsH mutant was restricted in its recombination with the tsB and tsI mutants and the tsG and tsJ mutants failed to recombine at detectable levels at nonpermissive temperature. The mutants of groups F-J did not complement, or did so inefficiently, and interfered with the growth of wild type at both permissive and nonpermissive temperatures. The growth properties of the mutants of groups F-J are described.

Animals↗

PubMed1997

Genetic characterization of the rotavirus nonstructural protein, NSP4.

The rotavirus nonstructural protein NSP4 plays a role in viral assembly by acting as an intracellular receptor for single-shelled particles and assisting in the translocation of these particles across the endoplasmic reticulum. Recently, NSP4 has been implicated in rotavirus virulence and is thought to act as an enterotoxin which triggers chloride secretion by a calcium-dependent signal transduction pathway. Limited sequence analysis of NSP4 shows a well-conserved protein. To define the extent of sequence variation in the gene coding for NSP4, we have sequenced this gene from nine human rotavirus strains. These data and the analysis of additional human strains and various animal rotaviruses (bovine, simian, equine, and porcine) by Northern blot hybridization suggested that three NSP4 genotypes were present among rotavirus strains. A correlation between NSP4 genotype and VP6 subgroup was also implied. Two different NSP4 genes (which encoded distinct types of NSP4 proteins) were found among standard human rotaviruses and in strains circulating in the local community and these showed homology to cognate genes in some animal strains.

Amino Acid Sequence↗

PubMed1989

Genetic characterization of the mei-41 locus in Drosophila melanogaster.

The mutagen-sensitive mutant mus(1) 104D1 of Drosophila melanogaster maps to a position on the X chromosome very close to the meiotic mutant mei-41D5. Both mutants have been characterized as mutagen-sensitive and defective in post-replication repair. In the present report we show by complementation studies that mus(1) 104 and mus(1) 103 are allelic with mei-41. In addition, two reported alleles of mus(1) 104 lie between the mei-41 alleles A10 and D5. The size of the mei-41 locus is estimated to be about 0.1 centimorgans (cM). Because several alleles of mei-41 have been shown to reduce recombination and increase meiotic chromosome loss and nondisjunction, mus(1) 104D1 females were examined for defects in meiosis. Although there was no evidence for reduced recombination on the second chromosome in homozygous mus(1) 104D1 females, heterozygous mus(1) 104D1/mei-41D5 and mus(1) 104D1/deficiency females showed reduced levels of recombination. However, there was no evidence of an increase in nondisjunction in these females.

Alleles↗

PubMed2003

Genetic characterization of the styrene lower catabolic pathway of Pseudomonas sp. strain Y2.

Pseudomonas sp. strain Y2 is a styrene-degrading bacterium, which initiates the catabolism of this compound via its transformation into phenylacetate by the sequential oxidation of the vinyl side chain. The styrene upper catabolic gene cluster (sty genes) had been localized in a 9.2-kb chromosomal region. This report describes the isolation, sequencing and analysis of an adjacent 20.5-kb chromosomal region that contains the genes of the styrene lower degradative pathway (paa genes), which are involved in the transformation of phenylacetate into aliphatic compounds that can enter the Krebs cycle. Hence, Pseudomonas sp. strain Y2 becomes the first microorganism whose entire styrene catabolic cluster has been completely characterized. Analysis of the paa gene cluster has revealed the presence of 17 open reading frames as well as gene duplications and gene reorganizations that are absent in other phenylacetate catabolic clusters described so far. The functionality of these genes has been proved by means of both complementation experiments on Pseudomonas putida mutants and in vitro enzymatic assays. Moreover, a DNA cassette encoding the whole styrene lower pathway has been constructed and has been used to expand the ability of Pseudomonas strains to degrade phenylacetic acid. For the first time, two functional phenylacetate-CoA ligases have been identified in an aerobic phenylacetic acid degradation pathway. Although the upper and lower styrene catabolic clusters are adjacent in the Pseudomonas sp. strain Y2 chromosome, their particular base composition and codon usage suggest a distinct evolutionary history.

Base Sequence↗

PubMed1999

Ribonucleotide reductase (RNR) of Corynebacterium glutamicum ATCC 13032--genetic characterization of a second class IV enzyme.

Ribonucleotide reductases (RNRs) encoded by nrd (nucleotide reduction) genes are unique enzymes providing the DNA precursors in all living organisms and several viruses. The designation of four classes of RNRs reflects their use of diverse metallo-cofactors. Using oligonucleotide primers derived from conserved domains of the primary structure of known NrdA and NrdE proteins, an internal 938 bp fragment of the nrdE gene was amplified from genomic DNA of Corynebacterium glutamicum. With this PCR product a 4.36 kb fragment was identified and cloned containing the nrdHIE genes of C. glutamicum. A probe derived from nrdF2 of Mycobacterium tuberculosis allowed the cloning and sequencing of the nrdF gene located 3.1 kb further downstream, encoding the small subunit of the C. glutamicum RNR. Conjugative introduction of nrdE from C. glutamicum complemented thermosensitive mutants of Corynebacterium ammoniagenes which had a defective catalytic subunit of the Mn-RNR. The authors provide arguments for allocation of the C. glutamicum NrdEF proteins to class IV in the RNR classification scheme of Stubbe & van der Donk (1995) [Chem Biol 2, 793-801].

Amino Acid Sequence↗

PubMed1988

Temperature-sensitive mutants of mouse hepatitis virus type 3 (MHV-3): isolation, biochemical and genetic characterization.

Mouse hepatitis virus 3 (MHV-3) is highly hepatotropic in sensitive mice. Temperature-sensitive mutants (ts mutants) induced by N-methyl-N'-nitrosoguanidine and 5-fluorouracil were isolated. Twelve mutants which were able to induce the formation of syncytia at 33 degrees C but not at the restrictive temperature (39.5 degrees C) were selected for detailed study. No viral RNA synthesis was detected after infection at the restrictive temperature with six of the mutants (RNA-) whereas six others were RNA+, although they displayed RNA synthesis which was generally reduced. No differences have been detected in the size of the genome or the viral-intracellular RNA species found in wild type virus or ts mutant infected cells at permissive temperature. The pattern of virus-induced proteins analyzed after immunoprecipitation by SDS-PAGE was similar in wild type virus and RNA+ mutant infected cells at 39.5 degrees C. Complementation experiments between ts mutants enabled us to distinguish five groups. Three of the groups contained RNA- mutants and two of them RNA+. Plaques made by mutants in one group displayed characteristic features that distinguished them from the wild type.

Animals↗

PubMed1981

Isolation and genetic characterization of toxin-deficient mutants of Pseudomonas aeruginosa PAO.

Two independently derived, exotoxin A-deficient (Tox- phenotype), nitroso-guanidine-induced mutants of Pseudomonas aeruginosa PAO1 were isolated by using sensitive immunological assays. One mutant, designated PAOT10, was detected as a colony which failed to produce a halo of immunoprecipitation in an antiserum-agar assay. The other mutant (PAOT20) was independently isolated and was detected by a negative reaction in a staphylococcal coagglutination assay with protein A-containing staphylococci and affinity-purified antibodies. Both mutants produced parental levels of extracellular protein. However, whereas the qualitative and quantitative compositions of proteins produced by PAOT20 were indistinguishable from those of the parental strain as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and measurement of extracellular protease, there were marked differences between PAOT10 and the parental strain. The mutation in PAOT10 (tox-1) as mapped by linkage analysis was located between trp-6 and proA. In contrast, linkage analysis and cotransduction placed the mutation in PAOT20 (tox-2), very near trp-6. Data are presented which suggest that tox-1 and tox-2 are regulatory loci.

ADP Ribose Transferases↗

PubMed1989

Genetic characterization of Bacillus subtilis odhA and odhB, encoding 2-oxoglutarate dehydrogenase and dihydrolipoamide transsuccinylase, respectively.

The 2-oxoglutarate dehydrogenase complex consists of three different subenzymes, the E1o (2-oxoglutarate dehydrogenase) component, the E2o (dihydrolipoyl transsuccinylase) component, and the E3 (dihydrolipoamide dehydrogenase) component. In Bacillus subtilis, the E1o and E2o subenzymes are encoded by odhA and odhB, respectively. A plasmid with a 6.8-kilobase-pair DNA fragment containing odhA and odhB was isolated. Functional E1o and E2o are expressed from this plasmid in Escherichia coli. Antisera generated against B. subtilis E1o and E2o expressed in E. coli reacted with antigens of the same size from B. subtilis. The nucleotide sequence of odhB and the terminal part of odhA was determined. The deduced primary sequence of B. subtilis E2o shows striking similarity to the corresponding E. coli protein, which made it possible to identify the lipoyl-binding lysine residue as well as catalytic histidine and aspartic acid residues. An mRNA of 4.5 kilobases hybridizing to both odhA and odhB probes was detected, indicating that odhA and odhB form an operon.

Acyltransferases↗