Complementation of a nonmotile flaB mutant of Borrelia burgdorferi by chromosomal integration of a plasmid containing a wild-type flaB allele.

With the recent identification of antibiotic resistance phenotypes, the use of reporter genes, the isolation of null mutants by insertional inactivation, and the development of extrachromosomal cloning vectors, genetic analysis of Borrelia burgdorferi is becoming a reality. A previously described nonmotile, rod-shaped, kanamycin-resistant B. burgdorferi flaB::Km null mutant was complemented by electroporation with the erythromycin resistance plasmid pED3 (a pGK12 derivative) containing the wild-type flaB sequence and 366 bp upstream from its initiation codon. The resulting MS17 clone possessed erythromycin and kanamycin resistance, flat-wave morphology, and microscopic and macroscopic motility. Several other electroporations with plasmids containing wild-type flaB and various lengths (198, 366, or 762 bp) of sequence upstream from the flaB gene starting codon did not lead to functional restoration of the nonmotile flaB null mutant. DNA hybridization, PCR analysis, and sequencing indicated that the wild-type flaB gene in nonmotile clones was present in the introduced extrachromosomal plasmids, while the motile MS17 clone was a merodiploid containing single tandem chromosomal copies of mutated flaB::Km and wild-type flaB with a 366-bp sequence upstream from its starting codon. Complementation was thus achieved only when wild-type flaB was inserted into the borrelial chromosome. Several possible mechanisms for the failure of complementation for extrachromosomally located flaB are discussed.

Genetic variability and linkage disequilibrium within the HLA-DP region: analysis of 15 different populations.

In order to understand the forces governing the evolution of the genetic diversity in the HLA-DP molecule, polymerase chain reaction (PCR)-based methods were used to characterize genetic variation at the DPA1 and DPB1 loci encoding this heterodimer on 2,807 chromosomes from 15 different populations including individuals of African, Asian, Amerindian, Indian and European origin. These ethnically diverse samples represent a variety of population substructures and include small, isolated populations as well as larger, presumably admixed populations. Ten DPA1 and 39 DPB1 alleles were identified and observed on 87 distinct DP haplotypes, 34 of which were found to be in significant positive linkage disequilibrium in at least one population. Some haplotypes were found in all ethnic groups while others were confined to a single ethnic group or population. Strong positive global linkage disequilibrium (Wn) between DPA1 and DPB1 was present in all 15 populations. The African populations displayed the lowest values of Wn whereas the Amerindian populations displayed near absolute disequilibrium. Analysis of the distribution of haplotypes using the normalized deviate of the Ewens-Watterson homozygosity statistic, F, suggests that DP haplotypes encoding the functional heterodimer are subject to much lower degrees of balancing selection than other loci within the HLA region. Finally, neighbor joining tree analyses demonstrate the power of haplotype diversity for inferring the relationships between the different populations.

[Study of the most often encountered allele of the HLA-B27 gene in Tuvinian and Russian inhabitants of Western Siberia]. / Izuchenie naibolee chasto vstrechaiushchikhsia allelei gena HLA-B27 u tubintsev i russkikh zhitelei zapadnoi Sibiri.

HLA-B27 gene frequencies and allelic polymorphism were studied in two Siberian ethnic groups: Russians from Novosibirsk (western Siberia) and Tuvinians from Kyzyl (southern Siberia). The HLA-B27 frequencies were determined by means of serologic typing of HLA antigens in 198 Tuvinians and 288 Russians. Molecular typing was performed via hybridization of oligonucleotide probes with amplified DNAs obtained from 30 HLA-B27-positive Russians and 11 HLA-B27-positive Tuvinians. The HLA-B27 gene frequencies in Tuvinians and Russians were 5.5 and 10.4%, respectively. Molecular variants of the HLA-B27 gene were studied in Tuvinians for the first time. The proportions of the HLA-B2705 and HLA-B2704 alleles were found to be 64 and 36%, respectively, in the population studied. The presence of the HLA-B2704 allele indicates a Mongoloid origin of Tuvinians. In the Russian population of Novosibirsk, the HLA-B2704 allele was not found, whereas the proportions of the HLA-B2705 and HLA-B2702 alleles were 76.2 and 23.8%, respectively, which is characteristic of Caucasoid populations.

[The use of internal controls of different lengths in the detection of Chlamydia trachomatis DNA by the polymerase chain reaction method]. / Ispol'zovanie vnutrennikh kontrolei razlichnoi dliny pri detektsii DNK Chlamydia trachomatis metodom polimeraznoi tsepnoi reaktsii.

To detect C. trachomatis DNA, the polymerase chain reaction (PCR) with the use of primers corresponding to variable sites of rRNA gene 16S was carried out. As the positive control of the reaction, the amplification fragment of gene 16S of rRNA, cloned in the plasmid vector and having the length of 530 nucleotide pairs (n.p.), was used. On its basis 2 kinds of the internal control of the reaction were obtained with the deletion of 110 n.p. (pMOS-Chl420) and the insertion of 930 n.p. (pMOS-Chl1460) within the cloned amplification fragment. The study revealed that the addition of the DNA of pMOS-Chl420 or pMOS-Chl1460 into the reaction mixture did not affect the sensitivity of PCR (0.02 pg of bacterial DNA in the sample) in the detection of C. trachomatis DNA isolated both from the culture of bacterial cells and from clinical samples. But in some cases of the amplification of the DNA of internal control pMOS-Chl420, but not pMOS-Chl1460, was observed in the presence of DNA obtained from clinical samples. It was supposedly linked with a higher sensitivity of Taq DNA-polymerase to the action of inhibitors in the synthesis of high-molecular DNA fragments. The observed high frequency of the inhibition (17%) of PCR makes it expedient to carry out this reaction with the use of the internal control.

[Association of TAP1/TAP2 gene polymorphism with inflammatory urogenital diseases combined with joint diseases]. / Assotsiirovannost' polimorfizma genov TAP1/TAP2 s vospalitel'nymi zabolevaniiami urogenital'nogo trakta, sochetaiushchimisia s porazheniiami sustavov.

AIM: To study association of TAP1/TAP2 gene polymorphism with urogenital infections combined with joint lesions. MATERIALS AND METHODS: Of 139 patients examined 45 ones had inflammation caused by Chlamydia trachomatis (17 had joint disease), 42 had Mycoplasma hominis infection (joint lesions in 17 cases). Method of amplification was used on the basis of specific primers (ARMS). RESULTS: Patients infected with C. trachomatis significantly more frequently had allele TAP1-02011 and TAP2-0201 (RR = 18.5, p < 0.01 and RR = 4.61, p < 0.05, respectively). Joint lesion in patients with chlamydial infection was associated with allele TAP1-02011 (RR = 11.92, p < 0.05). In mycoplasmosis association with joint lesions there is a significant link of joint syndrome with heterogeneous combination threonine/alanine in gene TAP2 position 565 (RR = 4.22, p < 0.05). CONCLUSION: The findings can be used for predicting the joint syndrome development in patients with urogenital infection.

[Frequency of the HLA-DP genes and the antigens of the HLA-A, -B, -Cw, and -DR loci in Tuvinians]. / Chastota genov HLA-DP i antigenov lokusov HLA-A, -B, -Cw. -DR u tuvintsev.

The results of DNA typing of the HLA-DPB1 gene among Tuvinians are described. The allele 0201 was found to be the most frequent (26.66%); then, in order of decreasing frequency, followed the alleles 0401 (26.42%), 0402 (13.99%), 0301 (8.55%), 0501 (7.77%), 0901 (5.16%), and 1301 (2.07%). The frequency of the allele 0501 in Tuvinians was significantly lower than that in the published data on allele distributions characteristic of two other Mongoloid populations, the Manchu and Japanese. The results of the serological typing of the loci HLA-A, -B, and -Cw of class I in the Tuva population were compared to the corresponding published data on ten Mongoloid populations of Central and Eastern Asia: Uigurs, Kazakhs, Mongols, Manchu, Tibetans, the Chinese of the northern and southern regions of China, Thais (inhabitants of Northwestern Indochina), Koreans, and Japanese. In Tuvinians, the most frequent antigens were HLA-A2, -A9, -B0, -B35, -B17, -Cw4, and -DR5. In the inhabitants of Tuva, the frequency of the antigens HLA-A1, -A9, -B17, -Cw2, and -Cw4 was significantly higher than in other Mongoloids, while that of the antigens HLA-A26 and -B22 was lower. This difference was greatest in the case of antigen HLA-B40: it a frequency of 43.4% was detected in it, while in individuals of Mongolian origin, the frequency of this antigen did not exceed 22.5%. Indices of genetic similarity (IGSs), calculated from the gene frequencies of the loci HLA-A, -B, and -Cw, suggested that the Tuvinians were most closely related to Kazakhs (IGS = 0.73), and least related to the Japanese and Uigurs (IGS = 0.66). Our data suggest that Tuvinians significantly differ from the compared populations of Central and Eastern Asia.

[Polymorphism of the HLA-DRB1 04 gene in inhabitants of Tuva]. / Polimorfizm gena HLA-DRB1 04 u zhitelei Tuvy.

Data on the HLA-DRB1 04 gene typing among Tuvinians from Kyzyl are presented. Experiments were conducted by using the hybridization of allele-specific oligonucleotides with the amplified fragments of high molecular-weight DNA and reverse hybridization. The HLA-DRB1 0401 allele was most frequent among DR4-positive individuals. The HLA-DRB1 0405 and HLA-DRB1 0403 alleles were found at somewhat lower frequencies. In one individual, the rare HLA-DRB1 0410 subtype differing from HLA-DRB1 0405 subtype by the substitution of glycine for valine in codon 86 was detected.

[The DNA typing of the HLA-B27 gene among individuals with joint lesions]. / DNK-tipirovanie gena HLA-B27 sredi lits s porazheniem sustavov.

To determine the HLA-B27 gene subtypes associated with joint diseases we examined 55 patients (35 with ankylosing spondylarthritis (AS), 13 with rheumatoid arthritis (RA), 5 with Reiter's syndrome, 2 with psoriatic arthritis). DNA amplification combined with sequence specific oligonucleotide hybridization were used to identify six HLA-B27 gene alleles. In the AS patients group the frequency of HLA-B2705 variant was found to be different significantly from that in healthy West Siberia Caucasians and distribution of HLA-B27 gene allele variants among AS patients in East Russia differed from that in West regions of Russia. DNA typing data can be useful together with other criteria for diagnosis of AS in a early onset.

[Frequency distribution of HLA phenotypes, haplotypes and alleles in ethnic groups of Taimyr and Chukotka]. / Rasprostranenie chastot fenotipov, gaplotipov, allelei HLA v étnicheskikh gruppakh Taimyra i Chukotki.

HLA typing data of two little populations living in Tajmyr peninsula (Dolgans and Nganasans) and Chukotka peninsula (Chukchas and Chuvantses) were presented. Our data were compared with the International standard distribution of class I HLA antigens in orients and its distribution in orients who live in the Asian part of Russia. Indexes of genetic distribution and genetic likeness were calculated.

[Features of the distribution of alleles of the HLA-DRB1 04 and HLA-DQB1 03 genes among healthy people of European origin in Western Siberia]. / Osobennosti raspredeleniia allelei genov HLA-DRB1 04 i HLA-DQB1 03 sredi zdorovykh lits Evropeiskogo proiskhozhdeniia v Zapadnoi Sibiri.

The allelic HLA-DRB1 04 and HLA-DQB1 03 polymorphism in caucasians living among the West Siberia Mongoloid aborigenes was studied. As a result of our studies, it was shown that the HLA-DRB1 0403/07 predominates and HLA-DRB1 0404/08 is absent in the Russian population of West Siberia, in contrast to those among Caucasians living in West Europe and North America. The frequencies of HLA-DQB1 03 alleles are similar to those observed among the all Caucasians. Gametic association HLA-DR4 - HLA-DQw was found for the first time in Caucasians of West Siberia.