Six genes (nikA, nikB, nikD, nikE, nikF, and nikG) from Streptomyces tendae Tü901 were identified by sequencing the region surrounding the nikC gene, which encodes L-lysine 2-aminotransferase, previously shown to catalyze the initial reaction in the biosynthesis of hydroxypyridylhomothreonine, the peptidyl moiety of the peptidyl nucleoside antibiotic nikkomycin. These genes, together with the nikC gene, span a DNA region of 7.87 kb and are transcribed as a polycistronic mRNA in a growth-phase dependent manner. The sequences of the deduced proteins NikA and NikB exhibit significant similarity to those of acetaldehyde dehydrogenases and 4-hydroxy-2-oxovalerate aldolases, respectively, which are involved in meta-cleavage degradation of aromatic hydrocarbons. The predicted NikD gene product shows sequence similarity to monomeric sarcosine oxidases, and the deduced NikE protein belongs to the superfamily of adenylate-forming enzymes. The nikF gene and the nikG gene encode a cytochrome P450 monooxygenase and a ferredoxin, respectively. Disruption of any of the genes nikA, nikB, nikD, nikE and nikF by insertion of a kanamycin resistance cassette abolished formation of the biologically active nikkomycins I, J, X, and Z. The nikA, nikB, nikD, and nikE mutants accumulated the nucleoside moieties nikkomycins Cx and Cz. In the nikD and nikE mutants nikkomycin production (nikkomycins I, J, X, Z) could be restored by feeding with picolinic acid and hydroxypyridylhomothreonine, respectively. The nikF mutant exclusively produced novel derivatives, nikkomycins Lx and Lz, which contain pyridylhomothreonine as the peptidyl moiety. Our results indicate that the nikA, nikB, nikD, nikE, nikF, and nikG genes, in addition to nikC, function in the biosynthetic pathway leading to hydroxypyridylhomothreonine, the putative activities of each of their products are discussed.
In addition to coding region polymorphism, allele-specific variation in the upstream regulatory region of the HLA-DQB1 gene has been detected. Reporter gene assays and transfection studies have indicated that HLA-DQB1 promoter polymorphism may be of functional significance. The aim of this study was to utilize real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for allele-specific quantification of HLA-DQB1 expression and to analyze cell-specific HLA-DQB1 expression in vivo. For the allele-specific quantification of DQB1 gene products, a real-time RT-PCR set of primer pairs (n ¼ 27) and probes (n ¼ 5) targeting exon 2 variability was established. The robustness and integrity of the assay system were confirmed by using recombinant DQB1 exon 2 plasmid clones as active exogenous controls. Sensitivity and reproducibility were assessed by serial dilution and allelic mixing analyses. In application to the study of allele-specific expression of DQB1 gene products during cytokine-driven maturation of monocyte-derived dendritic cells, differential patterns of allelic expression in heterozygous individuals were observed for DQB1*0301, compared to DQB1*0501 and DQB1*0602. At maximum, 1.9-fold (*0301/*0501) and 2.5-fold (*0301/*0602) higher induction was seen for DQB*0301. In conclusion, HLA-DQB1 expression can be analyzed by real-time RT-PCR suitable for cell-and allele-specific detection of HLA-DQB1 transcripts in homo-and heterozygous combinations.
Background: Fuchs heterochromic cyclitis (FHC) is a chronic inflammatory eye disease, usually presenting as unilateral anterior uveitis. Up to date no disease susceptibility genes have been described for FHC. Methods: The allele frequency of HLA DRB1 and DQB1, polymorphisms of the tumour necrosis factor (TNF) α promoter region (–376, –308, –238), the promoter (–318), first exon (+49) and (AT)n repeat polymorphism of the cytotoxic T cell antigen 4 (CTLA4) gene were analysed in 44 FHC patients and 139 healthy controls. Results: The CTLA4 –318 C/T genotype was increased in FHC patients [odds ratio (OR) 3.0, 95% confidence interval (CI) 1.4–6.5], as well as long CTLA4 (AT)n microsatellite alleles with more than 16 AT repeats (OR 2.6, 95% CI 1.3–5.3). A trend towards the –308 G/A TNF-α genotype was found in the patient cohort, whereas no difference in HLA class II allele distribution was observed. Conclusion:CTLA4 but not TNF-α or HLA class II DRB1 and DQB1 may represent a candidate gene for disease susceptibility in FHC.
3491 Novel methods to quantify chimerism after allogeneic hematopoietic stem cell transplantation (HSCT) based on single-nucleotide polymorphism (SNP) specific real-time quantitative polymerase chain reaction (qPCR) require high amounts of input DNA. The applicability of these methods in cases where DNA quantity is limiting, however, is currently unclear. Here, we demonstrate that SNP typing performed with just 5ng of input DNA still allowed for the distinction of positive (mean ct 28.05) and negative (ct > 36) signals. In addition, we confirm the high informative value of a set of 19 SNP markers, with 12 markers exceeding 20% informativity in our population (n=74). Of interest, a four-fold reduction of input DNA for qPCR standard curves did not alter PCR efficiencies. Most importantly, in 7 out of 16 allogeneic HSCT patients who experienced disease relapse, a retrospective analysis using the SNP-qPCR method revealed re-appearance of recipient chimerism earlier (mean 95 days) compared to previous results obtained by a short tandem repeat (STR) specific PCR. Taken together our data clearly demonstrate that SNP-qPCR is more sensitive compared to the widely used STR-PCR even with reduced amounts of input DNA. We therefore recommend that SNP-qPCR is preferable to STR-PCR for chimerism analysis in cases where DNA quantity is a limiting factor. Disclosures: No relevant conflicts of interest to declare.
The HLA-C*07:315 allele is mostly identical to HLA-C*07:09 but has a non-synonymous substitution of C to G in exon 2.
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