New integron-associated gene cassette encoding a 3-N-aminoglycoside acetyltransferase

A fifth gene cassette containing an aacC gene, aacCA5, was found in an aacCA5-aadA7 cassette array in a class 1 integron isolated from a multiply drug resistant Salmonella enterica serovar Kentucky strain. The AacC-A5 or AAC(3)-Ie acetyltransferase encoded by aacCA5 is related to other AAC(3)-I enzymes and confers resistance to gentamicin.     

Analysis of the aac(3)-VIa gene encoding a novel 3-N-acetyltransferase.

Biochemical analysis (G. A. Papanicolaou, R. S. Hare, R. Mierzwa, and G. H. Miller, abstr. 152, Program Abstr. 29th Intersci. Conf. Antimicrob. Agents Chemother., 1989) demonstrated the presence of a novel 3-N-acetyltransferase in Enterobacter cloacae 88020217. This organism was resistant to gentamicin, and the MIC of 2'-N-ethylnetilmicin for it was fourfold lower than that of 6'-N-ethylnetilmicin, a resistance pattern which suggested 2'-acetylating activity. However, high-pressure liquid chromatography analysis demonstrated that the enzyme acetylated sisomicin in the 3 position. We have cloned the structural gene for this enzyme from a large (> 70-kb) conjugative plasmid present in E. cloacae. Subcloning experiments have localized the aac(3)-VIa gene to a 2.1-kb Sau3A fragment. The deduced AAC(3)-VIa protein showed 48% amino acid identity to the AAC(3)-IIa protein and 39% identity to the AAC(3)-VII protein. Examination of the 5'-flanking sequences demonstrated that the aac(3)-VIa gene was located 167 bp downstream of the aadA1 gene and was present in an integron. In addition, the aac(3)-VIa gene is also downstream of a 59-base element often seen in an integron environment. Primer extension analysis has identified a promoter for the aac(3)-VIa gene downstream of both the aadA1 gene and a 59-base element.     

Implications of phase variation of a gene (pgtA) encoding a pilin galactosyl transferase in gonococcal pathogenesis

The pilin glycoprotein (PilE) is the main building block of the pilus of Neisseria gonorrhoeae (gonococcus [GC]). GC pilin is known to carry a disaccharide O-glycan, which has an alphaGal attached to the O-linked GlcNAc by a 1-3 glycosidic bond. In this report, we describe the cloning and characterization of the GC gene, pilus glycosyl transferase A (pgtA), which encodes the galactosyl transferase that catalyzes the synthesis of this Gal-GlcNAc bond of pilin glycan. A homopolymeric tract of Gs (poly-G) is present in the pgtA gene of many GC strains, and this pgtA with poly-G can undergo phase variation (Pv). However, in many other GC, pgtA lacks the poly-G and is expressed constitutively without Pv. Furthermore, by screening a large number of clinical isolates, a significant correlation was observed between the presence of poly-G in pgtA and the dissemination of GC infection. Poly-G was found in pgtA in all (24 out of 24) of the isolates from patients with disseminated gonococcal infection (DGI). In contrast, for the vast majority (20 out of 28) of GC isolated from uncomplicated gonorrhea (UG) patients, pgtA lacked the poly-G. These results indicate that Pv of pgtA is likely to be involved in the conversion of UG to DGI.     

Detection of HNA-3a and -3b antibodies using transfected cell lines and recombinant proteins

BACKGROUND: HNA‐3 is a diallellic system located on choline transporter‐like protein 2 (CTL2), defined by a polymorphism at Amino Acid 154. HNA‐3a antibodies are of clinical importance in transfusion‐related acute lung injury but antibody detection requires labor‐intensive granulocyte isolation from HNA‐typed donors and the use of techniques such as the granulocyte agglutination test or granulocyte immunofluorescence test. Also, there is no commercial test for detection of HNA‐3 antibodies. STUDY DESIGN AND METHODS: HEK293 cells were transfected to generate stable cell lines expressing CTL2 fragments (Amino Acids 55‐230) and full‐length membrane bound CTL2 with HNA‐3a and ‐3b epitopes. Soluble fragments were used in enzyme‐linked immunosorbent assays to detect HNA‐3 antibodies. The cell lines expressing full‐length proteins were trypsin treated to remove HLA antigens and frozen at ?80°C. Thawed cells were then used to detect HNA‐3 antibodies by flow cytometry. RESULTS: Glycosylated and soluble CTL2 fragments were correctly recognized by 15 of 31 anti‐HNA‐3a sera and by both available anti‐HNA‐3b sera. Twenty‐one anti‐HLA sera reacted variably with untreated cell lines expressing full‐length CTL2. After trypsin treatment of the cell lines, reactivity with HLA antisera was abrogated and all 31 anti‐HNA‐3a and two anti‐HNA‐3b sera bound to the corresponding cell line. CONCLUSION: Whereas soluble, glycosylated CTL2 fragments cannot be used for the detection of HNA‐3 antibodies, the HEK293 cells expressing full‐length CTL2 proteins were useful in the detection of HNA‐3 antibodies even in the presence of HLA antibodies. Moreover, the cell lines can be stored for at least 6 months before use.     

Genetic variation within a parthenogenetic lineage

A clone of the grain aphid Sitobion avenae F. was maintained parthenogenetically over thirty-two generations (n= 344) in a constant environment: a new generation being set up by a female selected at random from the preceding generation. Genomic DNA from individual aphids was screened for genetic stability using RAPD-PCR with a previously tested ten-mer primer. A putative germ-line mutation was noted in generation 14 and somatic mutations were noted in generations 12, 25, 27 and 29. There were no differences in the RAPD-PCR profiles of winged and wingless morphs and samples tested for symbiotic DNA. No endobiotic fungal organism was associated with the clone. Southern blotting and hybridization studies indicated that band additions were of aphid origin. However, the RAPD-PCR profiles of the germ-line and somatic mutation samples were unique from other aphid clones cultured during the experimental period. This paper documents discernible genetic changes occurring within an animal clonal lineage over time and impacts on the consequences this may have for clonal systems.     

Genetic variation in interleukin-10 gene and risk of oral cancer

BACKGROUND: Common genetic variants in immune and inflammatory response genes can affect the risk of developing oral cancer. Interleukin-10 (IL-10) is an immunosuppressive cytokine which may facilitate development of cancer by supporting tumor escape from the immune response. Inter-individual variations in IL-10 production were genetically contributed to polymorphisms within IL-10 promoter region. We determined whether single nucleotide polymorphisms (SNPs) at positions -1082 A/G (rs1800870), -819 T/C (rs1800871) and -592 A/C (rs1800872) in the IL-10 gene promoter were involved in predisposing an individual to oral cancer. METHODS: We analyzed 3 SNPS of IL-10 gene promoter in 280 patients with oral cancer and 300 age and sex matched controls in a Chinese population, using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) strategy. RESULTS: There were significant differences in the genotype and allele distribution of -1082 A/G (rs1800870) polymorphism of the IL-10 gene among cases and controls. The -1082 G alleles carriers were associated with a significantly increased risk of oral cancer compared with the non-carriers (OR=1.821, 95% CI, 1.329-2.496, P<0.001). Haplotype analysis revealed that the GCC haplotype (defined by SNPs at positions -1082, -819 and -592) of IL-10 gene conveys the highest risk for oral cancer compared with the ATA haplotype (OR=1.716; 95% CI, 1.230-2.395; P=0.001). CONCLUSION: IL-10 gene promoter -1082 A/G (rs1800870) polymorphism, and its haplotype are significantly associated with the risk of oral cancer. Our data suggests that IL-10 gene plays an important role in the development of oral cancer.     

Full-length recombinant choline transporter-like protein 2 containing arginine 154 reconstitutes the epitope recognized by HNA-3a antibodies

BACKGROUND: Recent reports have shown that the HNA‐3a leukocyte antigen, a target for antibodies that cause severe transfusion‐related acute lung injury, correlates with an arginine 154 (rather than glutamine) polymorphism in choline transporter–like protein 2 (CTL2) but did not show directly that R154 determines HNA‐3a. CTL2 peptides containing R154 are recognized by only half of HNA‐3a antibodies studied to date. Constructs that react with all HNA‐3a antibodies are needed to fully define the HNA‐3a epitope. STUDY DESIGN AND METHODS: HEK293 cells were transfected with cDNA encoding full‐length CTL2 linked to green fluorescent protein (GFP). Transfectants were selected for GFP expression and tested with antibodies specific for HNA‐3a and ‐3b. RESULTS: Each of 20 HNA‐3a antibodies reacted preferentially with HEK293 cells expressing the R154 CTL2 construct. An HNA‐3b antibody reacted only with CTL2 (Q154). CONCLUSIONS: These findings provide direct evidence that R154 in the context of full‐length CTL2 is both necessary and sufficient to create the HNA‐3a epitope but suggest that posttranslational modifications of the protein, for example, S‐S bonds or addition of glycans, are necessary for recognition of HNA‐3a by many antibodies. This could complicate development of an assay for large‐scale screening of blood donors to detect anti‐HNA‐3a.     

Molecular characterization of the gene encoding a new AmpC beta-lactamase in a clinical strain of acinetobacter genomic species 3

A presumptive chromosomal cephalosporinase (pI, 9.0) from a clinical strain of Acinetobacter genomic species 3 (AG3) is reported. The nucleotide sequence of this beta-lactamase shows for the first time the gene encoding an AmpC enzyme in AG3. In addition, the biochemical properties of the novel AG3 AmpC beta-lactamase are reported     

Genetic risk factors and ischaemic cerebrovascular disease: role of common variation of the genes encoding apolipoproteins and angiotensin-converting enzyme

DNA polymorphisms in genes encoding apolipoproteins (apo) A-I, C-III, B and E and angiotensin-converting enzyme (ACE) have been proposed to be associated with the risk of coronary artery disease (CAD). We studied whether the same genetic markers would also be associated with the occurrence and extent of atherosclerosis in cervical arteries. DNA samples from 234 survivors of stroke or a transient ischaemic attack aged 60 years or less were examined. The presence of atherosclerosis was assessed using aortic arch angiograms. The SstI polymorphism of apoA-I/C-III gene locus, the Xbal polymorphism of apoB gene, common apoE phenotypes and the insertion/deletion polymorphism of the ACE gene were analysed. The allele frequencies of the apoA-I/C-III, apoB, apoE or ACE gene did not differ between the groups with (n = 148) or without (n = 85) cervical atherosclerosis. However, when patients with at least one apoE4 allele and one X2 allele of apoB were combined and compared with those without either of them (E2E3 or E3E3 and X1X1), a significant association with the presence of cervical atherosclerosis was found (P = 0.03). The patients having the E2E3 phenotype had a significantly elevated serum triglyceride level compared with those with the E3E3 phenotype (P = 0.03). Serum high-density lipoprotein (HDL) cholesterol was lower in the patients with the E2E3 phenotype than in those with the E3E3 and E3E4 (P = 0.01 and P = 0.06, respectively). The apoB or ACE genotypes were not significantly associated with serum lipid or lipoprotein levels. There was no association between the ACE gene polymorphism and the occurrence of hypertension. In conclusion, the interaction of common apoB and apoE alleles may increase the risk of atherosclerosis in cervical arteries.     

Gene frequencies of the HNA-4a and -5a neutrophil antigens in Brazilian persons and a new polymerase chain reaction-restriction fragment length polymorphism method for HNA-5a genotyping

BACKGROUND: The HNA-4a (Mart) and HNA-5a (Ond) antigens are polymorphic variants of alpha(M) (CD11b) and alpha(L) (CD11a) subunits of the beta(2)-integrin, and are associated with single nucleotide polymorphisms (SNP) leading to amino acid dimorphisms. HNA-4a has been linked to alloimmune neonatal neutropenia, but the HNA-5a clinical significance is unclear. STUDY DESIGN AND METHODS: Using a polymerase chain reaction (PCR) with sequence-specific primers, the frequency of HNA-4a among 121 Brazilian blood donors and 114 Amazon Indians was determined. A PCR-restriction fragment length polymorphism (RFLP) method for HNA-5a genotyping was developed, and the gene frequencies of this antigen were determined among 123 blood donors and 114 Indians. To validate the genotyping method, the amplified DNA from six previously obtained samples (two of each genotype) was sequenced. RESULTS: The HNA-4a (+/+), HNA-4a (+/-), and HNA-4a (-/-) genotype frequencies of blood donors (0.686, 0.273, 0.041) and Indians (1.000, 0.000, 0.000) were different (p < 0.01). The frequencies of HNA-5a (+/+), HNA-5a (+/-), and HNA-5a (-/-) genotypes among blood donors (0.512, 0.399, 0.089) and Indians (0.746, 0.219, 0.035) also differed (p < 0.01). Sequencing demonstrated concordance with PCR-RFLP genotyping in all six evaluated samples. CONCLUSION: Comparing to another populations, Brazilians present a higher frequency of HNA-4a-negative allele, suggesting that Brazilians would be more susceptible to HNA-4a alloimmunization. Moreover, the distribution of the HNA-4 alleles observed in Amazon Indians is quite similar to that reported among Koreans. Besides that, a new effective and efficient HNA-5a genotyping technique is now available for population studies.     

Transposition of the gene encoding a TEM-12 extended-spectrum beta-lactamase.

An isolate of Klebsiella oxytoca from the blood culture of a child with leukemia was found to produce two beta-lactamases, at least one of which conferred resistance to ceftazidime. Genes encoding both enzymes were located on a single self-transmissible 100-kb plasmid, pOZ201. This plasmid was introduced into Escherichia coli UB5201 (pACYC184), and the gene encoding one beta-lactamase was transposed onto plasmid pACYC184 by exploiting a gene dosage effect. The transposable gene was found to encode a TEM-12 enzyme as determined by nucleotide sequencing. This gene was subsequently transposed onto plasmid pUB307. The transposable element encoding the TEM-12 enzyme has been designated Tn841. Both plasmids pACYC184::Tn841 and pUB307::Tn841 were shown to encode a beta-lactamase with the same isoelectric point and substrate profile as the TEM-12 beta-lactamase. Transposon Tn841, at approximately 7 kb, is larger than TnA (4.8 kb) and transposes at a lower frequency. Although it produced a resolvase which can complement the resolvase of Tn3, its transposase function was not able to complement the transposition of a TnA element which lacked transposase. The occurrence of a gene encoding an extended-spectrum beta-lactamase on a transposable element in a clinically significant bacterium is potentially a cause for concern for the spread of resistance to the extended-spectrum cephalosporins.     

Characterization of the Drosophila melanogaster retinin gene encoding a cornea-specific protein

Two-dimensional analysis of head extracts from Drosophila melanogaster identified the four eye-specific protein spots corresponding to the retinin protein. The retinin protein spots were specifically stained with phosphoprotein-specific dye, suggesting that the retinin protein undergoes post-translational modification by phosphorylation. Northern blot analysis showed that the retinin gene begins to be expressed during the late stage of puparium formation during development. Analysis of the N-terminal sequence and expression of the retinin gene in S2 suggest that retinin is a secretory protein. Transgenic flies with knockdown expression of the retinin gene by RNA interference (RNAi) were established. However, no significant phenotypic changes in eye structure or phototransduction were observed in the transgenic flies. Western blot analysis and immunohistochemical studies of D. melanogaster eyes suggest that retinin is a cornea-specific protein.     

Cloning and characterization of the Pseudomonas aeruginosa pbpB gene encoding penicillin-binding protein 3.

Clones containing the pbpB gene which encodes penicillin-binding protein (PBP) 3 of Pseudomonas aeruginosa were detected by hybridization by PCR amplification with primers based on the conserved sequences of high-molecular-weight PBPs. The translated amino acid sequence demonstrated 45% identity and had a total of 66% conserved amino acids relative to the Escherichia coli PBP3. The pbpB gene was located upstream of a gene homologous to the E. coli murE gene, which encodes uridine diphosphate-N-acetyl muramic acid-tripeptide synthetase. The overexpressed pbpB gene product reacted with 3H-penicillin G and had an apparent molecular weight of 60,000.     

Genetic variation of FUT3 in Ghanaians, Caucasians, and Mongolians

BACKGROUND: The FUT3 gene regulates the expression of Lewis blood group antigens. Several lines of evidence suggest association between expression of these antigens and Helicobacter pylori infection or susceptibility to cardiovascular diseases. Single-nucleotide polymorphisms (SNPs) responsible for the Lewis-negative phenotype have been reported, but systematic sequence analyses have not yet been performed.
STUDY DESIGN AND METHODS: The polymerase chain reaction product containing the whole FUT3 coding region (1086 bp) was sequenced in three human populations, Ghanaians (n = 106), Caucasians (n = 100), and Mongolians (n = 50). Some haplotypes were determined by sequencing cloned inserts, and the other haplotypes were inferred by the free software program PHASE. Also examined was the functional significance of several newly identified SNPs by a transient expression study.
RESULTS: Thirty-two SNPs were found, including 15 novel SNPs, in the three populations. The functional impact of three nonsynonymous SNPs that occurred on the Lewis-positive alleles or weakly enzyme-inactivating alleles was examined. A transient expression study suggested that 478C>T and 968G>C are enzyme-inactivating mutations.
CONCLUSION: Sequence analysis identified many SNPs, but most of them are in complete linkage disequilibrium with previously reported SNPs responsible for the Lewis-negative phenotype. 13G>A, 59T>G, and 202T>C seem to be useful as tag SNPs for detection of Lewis-negative alleles in genotyping of Lewis blood groups and large-scale association studies with diseases in many populations.