Automated, solid-phase sequencing of DRB region genes using T7 sequencing chemistry and dye-labeled primers.
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Base Sequence↗
Biomedical subjects
M G Tilanus
Publications and source records attributed to M G Tilanus.
A complete exon 2 sequence of the HLA-DPA1*02012 allele.
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Alleles↗
Antisense oligonucleotides, a novel tool for the control of cytokine effects on human cartilage. Focus on interleukins 1 and 6 and proteoglycan synthesis.
OBJECTIVE: To prevent the negative effects of interleukin-1 (IL-1) and IL-1-induced IL-6 on cartilage proteoglycan (PG) synthesis, we used an antisense oligonucleotide (ASO) specific for IL-6 messenger RNA (mRNA) to inhibit IL-6 production. METHODS: Explants of human articular cartilage were cultured in the presence or absence of IL-6-ASO, IL-1, and exogenous IL-6. As metabolic parameters, cartilage production of IL-6 was determined in the B9 bioassay and PG as incorporation of 35SO4. RESULTS: The IL-6-ASO prevented IL-1-induced production of IL-6 in the cartilage explants, as well as IL-1-induced inhibition of PG synthesis. This inhibition was restored, despite the presence of IL-6-ASO, when exogenous IL-6 was added. A control ASO (not specific for IL-6 mRNA) was not effective. CONCLUSION: The IL-6-ASO used can penetrate the extracellular matrix of articular cartilage, enter the chondrocytes, and inhibit the IL-1-induced production of IL-6. Furthermore, IL-6-ASO can prevent the IL-1-induced inhibition of cartilage PG synthesis. The effect of exogenous IL-6 shows that IL-1 requires IL-6 for inhibition of PG synthesis.
Aged↗
Asian Indian HLA-DR2-, DR4-, and DR52-related DR-DQ genotypes analyzed by polymerase chain reaction based nonradioactive oligonucleotide typing. Unique haplotypes and a novel DR4 subtype.
We have employed a PCR-based nonradioactive technique using biotinylated SSOPs to define HLA-DR2-, 4-, DR51-, and DR52-associated DR-DQ genotypes in Asian Indian families. In the DR2 group, most haplotypes described by us in a previous study were confirmed by family analysis. Evidence for one additional haplotype was available in this study. The classic DRB1*1501- and DRB1*1502-associated caucasoid haplotypes occurred with an appreciable frequency in Asian Indians, but two of the DRB1*1601-associated Caucasoid haplotypes were absent. At least six unique and unusual DR2-associated genotypes were encountered. In the DR52 group, the three most common alleles are DRB1*0301, DRB1*1404, and DRB1*1101. The DR6-associated alleles were DRB1*1301, 1302, 1401, and 1404. A few unique haplotypes occurred with low frequency in this group. In the DR4 group, at least three unusual patterns of hybridization were noticed by family analysis. One of these appears to be a novel DR4 subtype upon sequencing. These results demonstrate that, besides HLA-DR2, appreciable complexity occurs in the DR4- and DR52-associated alleles among Asian Indians. The presence of unique DR-DQ haplotypes in addition to those found characteristically among Western Caucasians suggests that the Indian population provides valuable source of many HLA class II haplotypes.
Amino Acid Sequence↗
Mitochondrial control-region sequences in two shorebird species, the turnstone and the dunlin, and their utility in population genetic studies.
We determined the mitochondrial control-region sequences of five turnstones (Arenaria interpres) and three dunlins (Calidris alpina). Comparisons revealed that the central part (part II) is conserved relative to much more variable parts at the beginning (part I) and the end (part III). This pattern of sequence conservation is also found in the control regions of other vertebrates. The average sequence divergence between turnstone and dunlin was 21.8% for part I, 7.5% for part II, and 29.5% for part III. Within-species sequence divergence over the entire control region was much lower, at 0.9% for turnstones and 2.0% for dunlins. In both shorebird species, part III contains a repetitive sequence composed only of A and C nucleotides, which has not been found in the control regions of other birds. A survey of the part I sequences of 25 turnstones and 25 dunlins sampled around the world revealed that these species have very different population genetic structures. Dunlins are not only much more differentiated in their sequences but also have a strongly subdivided population genetic structure. Pleistocene vicariant events combined with strong natal philopatry and high mutation rates of the sequences are likely responsible for this population genetic subdivision. Conversely, part I sequences of turnstones are weakly differentiated and are geographically unstructured. We argue that this is not the result of global gene flow but that, instead turnstones have recently expanded from a refugial population that was bottlenecked.
Animals↗
Hypervariable-control-region sequences reveal global population structuring in a long-distance migrant shorebird, the Dunlin (Calidris alpina).
Hypervariable segments of the control region of mtDNA as well as part of the cytochrome b gene of Dunlins were amplified with PCR and sequenced directly. The 910 base pairs (bp) obtained for each of 73 individuals complete another of the few sequencing studies that examine the global range of a vertebrate species. A total of 35 types of mtDNA were detected, 33 of which were defined by the hypervariable-control-region segments. Thirty of the latter were specific to populations of different geographic origin in the circumpolar breeding range of the species. The remaining three types indicate dispersal between populations in southern Norway and Siberia, but female-mediated flow of mtDNA apparently is too low to overcome the effects of high mutation rates of the control-region sequences, as well as population subdivision associated with historical range disjunctions. A genealogical tree relating the types grouped them into five populations: Alaska, West Coast of North America, Gulf of Mexico, western Europe, and the Taymyr Peninsula. The Dunlin is thus highly structured geographically, with measures of mutational divergence approaching 1.0 for fixation of alternative types in different populations. High diversity of types within populations as well as moderate long-term effective population sizes argue against severe population bottlenecks in promoting this differentiation. Instead, population fragmentation in Pleistocene refuges is the most plausible mechanism of mtDNA differentiation but at a much earlier time scale than suggested previously with morphometric data.
Animals↗
Identification of two new nucleotide mutations (HPRTUtrecht and HPRTMadrid) in exon 3 of the human hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene.
Mutations in the X-linked hypoxanthine-guanine phosphoribosyl transferase gene (HPRT) result in deficiencies of HPRT enzyme activity, which may cause either a severe form of gout or Lesch-Nyhan syndrome depending on the residual enzyme activity. Mutations leading to these diseases are heterogeneous and include DNA base substitutions, DNA deletions, DNA base insertions and errors in RNA splicing. Identification of mutations has been performed at the RNA and DNA level. Sequencing genomic DNA of the HPRT gene offers the possibility of direct diagnostic analysis independent on the expression of the mature HPRT mRNA. We describe a Dutch and a Spanish family, in which the Lesch-Nyhan syndrome and a severe partial HPRT-deficient phenotype, respectively, were diagnosed. Direct sequencing of the exons coding for the HPRT gene was performed in both families. Two new exon 3 mutations have been identified. At position 16676, the normally present G was substituted by an A in the Dutch kindred (HPRTUtrecht), and led to an arginine for glycine change at residue 70. At position 16680, the G was substituted by a T in the Spanish family (HPRTMadrid); this substitutes a valine for glycine at residue 71. These new mutations are located within one of the clusters of hotspots in exon 3 of the HPRT gene in which HPRTYale and HPRTNew Haven have previously been identified.
Amino Acid Sequence↗
Assignment of HLA-DPB alleles by computerized matching based upon sequence data.
Routine HLA typing by serology has been supported by DNA or protein analysis of the respective molecules in cases when serologic typing was inconclusive or difficult to perform. DNA analysis by RFLP, SSO, or PCR amplification with SSP is a reliable tool for the identification of alleles. Because DNA sequences may now be determined routinely, we developed software based on sequence data from known sequences for allele assignment of polymorphic gene systems. We describe the assignment of HLA-DPB alleles based upon sequence data obtained after PCR amplification and subsequent sequencing of exon 2. Our software includes a database containing all known HLA-DPB sequences, which offers the possibility of analyzing heterozygous individuals by combinatorial comparison through all alleles and thus identifying the one or two alleles involved. Quality control of the sequence has been included by the alignment of constant regions of the sequence combined with related polymorphism of known polymorphic nucleotides and the identification of the positions crucial for the allele assignment. The ability to type for HLA-DPB alleles routinely by automatic sequence determination and subsequent automated analysis offers new perspectives for HLA-DNA typing.
Alleles↗
High-resolution HLA-DPB typing based upon computerized analysis of data obtained by fluorescent sequencing of the amplified polymorphic exon 2.
To differentiate 32 HLA-DPB alleles, conventional techniques such as serology and cellular typing are inadequate for high-resolution DPB typing. The most refined DNA typing until now is SSO typing and new selected oligonucleotides can be added to this system to distinguish new allele sequences. DNA sequencing, however, reveals directly the sequence information of all polymorphic HVRs and has the advantage of being independent from exon polymorphisms. We have developed a new DNA-based typing approach that is rapid, fully automated, and therefore suitable for routine typing. The system is based upon direct sequencing of amplified DNA with fluorescent-labeled primers. The designation of alleles is obtained by a comparison of all polymorphic positions in the determined sequence with all known allele sequences retained in a database along with their heterozygous combinations. Sequence data at both constant and polymorphic positions are used for quality control. In this study, the typing results of a panel of 91 previous SSO-typed DNA samples are described. After comparison with the SSO-typing results, we conclude that with this SBT system allele assignment is reliable. The method is easy to perform since both sequencing and assignment are automated. Furthermore, the system is easily applicable to other gene systems.
Base Sequence↗
Mapping of susceptibility to Marek's disease within the major histocompatibility (B) complex by refined typing of White Leghorn chickens.
The major histocompatibility (B) complex of a distinct commercial pure White Leghorn chicken line was characterized using serological, biochemical and restriction fragment length polymorphism (RFLP) typing. Line B chickens displayed a high recombination frequency within the B complex. Three recombinant haplotypes were identified. The influence of these haplotypes was determined in relation to the haplotypes B19 and B21 on their resistance to Marek's disease (MD) in an experimental infection with the virus. Offspring of sires with a recombinant haplotype in combination with B19 or B21, and dams, which were homozygous B19/B19 or B21/B21 were infected. The B type of the offspring had a significant effect upon survival. Animals with B complex types B21/B21, B134/B21 and B234/B21 were relatively resistant to MD (24-32% mortality), whereas B19/B19 birds were highly susceptible (68% mortality). Animals with a recombinant haplotype B19r21 (B-G21, B-F19) were equally susceptible to MD as birds with the complete B19 haplotype. In contrast to earlier publications, resistance was not inherited as a dominant trait. Apparently, B19 was associated with a dominant susceptibility. The gene(s) associated with the B complex and involved in resistance to MD were localized within the B-F/B-L region. However, the association with a presumably non-coding subregion of B-G could not be excluded.
Animals↗
A high recombination frequency within the chicken major histocompatibility (B) complex.
Chickens of a commercial pure White Leghorn line were typed for B-F and B-G by serological, biochemical and molecular biological methods. Amongst 287 typed animals of one particular line, three animals with recombinant haplotypes were identified. Compared to earlier reports this revealed a statistically significant (P < 0.05), tenfold higher recombination frequency in this chicken line.
Animals↗
Expression of T-cell receptor alpha and beta variable genes in normal and malignant human T cells.
A PCR method was developed to analyse each of 29 families of the T cell receptor V alpha gene and 20 families of the V beta gene at the mRNA level in heterogenous cell populations. All V alpha and V beta families were detectable in blood mononuclear cells from four of six healthy donors. In two donors only V alpha 22 was missing, and all other V alpha and V beta families were detected. V beta family expression was observed in T-leukaemic cell lines Jurkat, HSB, Molt-3 and Molt-4. In contrast, V alpha family expression was not detectable in any cell line except Jurkat cells. In T-cell malignancies (non-Hodgkin's lymphoma and mycosis fungoides), one or two V alpha and V beta families were detectable. Four of 10 cases investigated showed two V alpha transcripts and one V beta transcript. This fits with concepts in literature that allelic exclusion for the genes encoding alpha chains is not strictly required in the DNA rearrangement, or that this exclusion is a post-translational event. Using a limited series of antibodies to V beta gene family products, blood mononuclear cells from healthy donors were analysed by flow cytometry in a follow-up study. Two of four donors were rather stable in proportions of T cells expressing distinct V beta families, and two other donors showed variation in one or more families. When analysed on frozen tissue sections of normal lymph node and tonsil, there was no preferential location of lymphocytes expressing a distinct V beta gene family in different compartments (interfollicular area, follicle, or tonsillar epithelium).
Base Sequence↗
HLA-DR1 and rheumatoid arthritis in Israeli Jews: sequencing reveals that DRB1*0102 is the predominant HLA-DR1 subtype.
Rheumatoid arthritis (RA) is associated with the HLA-DR4 cellular subtypes Dw4 and Dw14 in Caucasians, with Dw15 in Japanese, and possibly with HLA-DR1 in Israeli Jews. Sequencing studies in Caucasians have shown that these molecules share a common amino acid sequence in the third hypervariable region of the DR molecule (AA 67-74: LLEQRRAA or LLEQKRAA), suggesting that this sequence is primarily associated with RA. An important argument in favor of this shared-epitope hypothesis has been the reported association between DR1 and RA in Israeli Jews. However, a later report did not confirm this association, and cellular typing showed that Israeli DR1 consists of three or more subtypes, suggesting that new subtypes might be present. Since no sequencing data on Israeli HLA-DR1 genes have been reported, we sequenced the first domain (AA 10-91) of the DRB1 gene in 12 DR1-positive Israeli RA patients, 5 healthy controls and a homozygous typing cell (HTC), defining the major Jewish cellular HLA-DR1 subtype. DRB1*0102 (DR1 Dw20) was found in 8 RA patients, 3 controls and the HTC "LVA". DRB1*0101 (DR1 Dw1) was found in 4 RA patients and 2 controls. No other DR1 subtypes were encountered. In all 20 DR1 haplotypes, the DRB1*0101 or 0102 allele was associated with DQA1*0101 and DQB1*0501, being identical to the Caucasian DR1 haplotypes. Thus, at the sequence level, we found no basis for the reported extensive cellular heterogeneity of DR1 in the Israeli population.(ABSTRACT TRUNCATED AT 250 WORDS)
Alleles↗
Aberrant oestrogen receptor species in human meningioma tissue.
Meningiomas are very rich in progestin receptors (PR) whereas oestrogen receptors (ER) are seldomly found and only at low concentrations. These tumours might possess an ER which is defective in oestrogen binding but still functional in stimulating oestrogen-responsive genes such as PR. In human meningiomas a polymerase chain reaction fragment including the DNA binding domain, the hinge region and the ligand binding domain of ER was amplified. The size of the fragment obtained was as expected from wild type mRNA sequences. Moreover, a variant, which was overexpressed in meningiomas, with a major deletion in exons 2-6 was detected.
Base Sequence↗
T-cell receptor V beta-family usage in primary cutaneous and primary nodal T-cell non-Hodgkin's lymphomas.
To evaluate whether the expression of T-cell receptor (TCR) V beta families in eight cases of malignant T-cell lymphomas took place in a preferential manner, we analyzed four cases of mycosis fungoides (MF), the most common form of primary cutaneous T-cell non-Hodgkin's lymphomas (NHL), and four cases of primary nodal T-cell NHL. The usage of V beta families in T-cell populations was investigated on mRNA that was transcribed to cDNA using a C beta primer and reverse transcriptase. Subsequently, the specific usage of the families was analyzed by polymerase chain reaction (PCR) using combinations of the selected C beta-oligonucleotide primer and one of the family-specific V beta primers. Peripheral blood lymphocytes from four healthy volunteers and 1 "reactive" lymph node served as a control and expressed all 20 V beta families tested for. In T-cell lines, with restricted V beta expression, and in three patients with advanced MF, only one or two V beta families were expressed at the mRNA level. In an early MF lesion this monoclonal expression was absent: several V beta families were expressed with a weak intensity. This may indicate either a polyclonal origin of MF, or that too few monoclonal neoplastic cells were present in the tissue specimen. In the four nodal T-cell NHL, only one family could be clearly distinguished, whereas some of the other V beta families showed only a weak expression. These latter families represent the reactive T-cell component in the nodal T-cell NHL. Both in nodal T-cell NHL and in MF there was no preferential expression of a particular V beta family. There was a good correlation between PCR data and the expression of V beta-family protein products observed by immunohistochemistry on tissue sections of the T-cell lymphomas. All T-cell lines, three cases of MF, and three cases of nodal T-cell NHL showed a rearrangement of the TCR beta chain on DNA level.
Base Sequence↗
T cell receptor V beta variable gene family expression in human peripheral blood lymphocytes at the mRNA and membrane protein level.
A polymerase chain reaction (PCR) was developed, using combinations of an oligonucleotide primer for a T cell receptor V beta gene family and one for the constant C beta gene segments, to assess the expression of each of 20 V beta gene families in RNA after reverse transcription into cDNA. The detection was done after agarose gel electrophoresis of PCR products and ethidium bromide staining. The positive identification of the PCR products was done by hybridization with a J beta oligonucleotide probe. For T cell lines, a signal was observed in the V beta 8 combination for Jurkat cells, V beta 5a in HSB cells, V beta 2 and V beta 12a in Molt-3 cells and V beta 2, V beta 5a and V beta 12a in Molt-4 cells. Using mixtures of RNA from different cell lines, the sensitivity of the method was in the range of 0.1-0.5%. In peripheral blood mononuclear cells from four donors, taken at three different occasions, all V beta families were detectable. The intensity of the PCR product varied between various V beta gene families. Flow cytometric analysis of blood mononuclear cells from the same donors with a restricted series of V beta gene family-specific antibodies also revealed the presence of all families. The approach to assess V beta gene family expression in heterogeneous populations opens the possibility to study T cell receptor variable gene expression in relation to physiology and pathologic processes.
Base Sequence↗
Detection of cytomegalovirus (CMV) in granulocytes by polymerase chain reaction compared with the CMV antigen test.
To compare the sensitivity and suitability of detection of active cytomegalovirus (CMV) infection by using monoclonal antibodies against CMV antigen (antigen test to detect antigenemia) and the polymerase chain reaction (PCR; to detect viral DNA) in granulocytes, 19 heart and 2 lung transplant recipients were closely monitored by these tests for at least 3 months after transplantation. All patients were CMV seropositive or had a seropositive donor. In total, 201 samples were tested; 46 were positive by both tests, 9 samples showed only antigenemia, 54 samples were positive by PCR only, and 102 samples were negative by both tests. PCR was positive earlier after transplantation in eight patients, whereas antigenemia was positive earlier after transplantation in one patient. In another four patients, both tests were positive at the same time. PCR was, on average, positive for a longer period of time. Discordant results showing a positive antigen test and a negative PCR were partly due to sampling error; some were positive by PCR on retesting. Samples which were negative by the antigen test and positive by PCR were taken at the beginning or at the end of an active CMV infection. In two patients, no active CMV infection was detected by the antigen test, cultures of urine and saliva, or serology, although PCR was positive for a long period of time in the two patients.
Antigens, Viral↗
A family with an apparent HLA-DR triplet: evidence for exchange of functional HLA-DR beta-genes between different haplotypes.
Serological and protein analysis showed that HLA-DR1 and DR2 short (s) segregated together as one haplotype, resulting in a HLA-DR triplet as found in a family study. Both DR1 and DR2 molecules were coordinately expressed and were shown to function as restriction elements in antigen presentation assays. This unique HLA-DR1, 2s haplotype was further studied by Southern blot analysis. Based upon well-known restriction fragment length polymorphisms for the involved gene sets, i.e. DR1 and DR2 along with the DR type from the other haplotype, the genes as identified by restriction fragment length polymorphism of the triplet could be established. Pseudogenes, which are included in the previously described gene sets of HLA-DR1 and DR2 are apparently lacking in the triplet. We therefore postulate that during an unequal crossing-over event the DR beta-pseudogene of DR2 could be exchanged by a functional DR1 beta-gene.
Crossing Over, Genetic↗