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Construction of a genomic map of Moraxella (Branhamella) catarrhalis ATCC 25238 and physical mapping of virulence-associated genes.

A physical genome map of the Moraxella catarrhalis type strain (ATCC 25238) has been constructed using pulsed field gel electrophoresis. Macrorestriction analyses of the genome of M. catarrhalis were performed by digestion with the restriction enzymes SmaI, NotI, and RsrII, which cleave the single circular chromosome into 9, 10, and 6 fragments, respectively. The chromosomal fragments generated by pulsed field gel electrophoresis were converted to a linkage map utilizing a combination of partial digestions, and cross-hybridizations. Moraxella catarrhalis, like a number of other respiratory pathogens, has a relatively small genome estimated at 1750 kilobase pairs or about 40% of the size of the Escherichia coli genome. The locations of the four ribosomal RNA operons (rrnLS) were determined by Southern hybridization and by digestion with I-CeuI endonuclease. A number of genes involved in virulence have been placed onto the physical map by Southern hybridization including those encoding the predominant outer-membrane proteins and the chromosomal gene encoding beta-lactamase.

Blotting, Southern↗

Mapping of centromeric regions on the molecular linkage map of rice (Oryza sativa L.) using centromere-associated sequences.

Genetic mapping of centromeres has been a challenge for plant geneticists. The objective of this study was to develop a new strategy for determining the locations of centromeric regions on genetic maps by mapping centromere-associated sequences, to make it possible to define the centromeric region of each chromosome as a single Mendelian locus on the molecular linkage map. Two DNA probes containing sequences specifically associated with the centromeres of grass species were used for genetic mapping. The centromere-associated sequences for all 12 rice chromosomes were mapped on the molecular map with either or both of the probes, and flanking molecular markers on one or both sides were localized 0 to 8 cM away. The map locations of the centromere-associated markers corresponded very well with the positions of centromeric regions determined previously using trisomic analyses for 11 of the 12 chromosomes. The precise mapping of the centromeric regions using these probes makes the molecular map a more complete and informative tool for genomic studies, which will facilitate studies of the structure and function of the rice centromeres. The simplicity of this technique, together with the fact that these probes are also associated with the centromeric regions in other grass species, may provide a general approach to the mapping of centromeric regions in the genomes of other cereal crops.

Base Sequence↗

AID binds to transcription-induced structures in c-MYC that map to regions associated with translocation and hypermutation.

Translocation and aberrant hypermutation of c-MYC are common in B-cell lymphomas. Activation-induced Cytidine Deaminase (AID) initiates switch recombination and somatic hypermutation in B cells by targeted deamination of transcribed genes. We show that transcription of the immunoglobulin S regions and c-MYC results in formation of similar DNA structures, 'G-loops', which contain a cotranscriptional RNA: DNA hybrid on the C-rich strand and single-stranded regions and G4 DNA on the G-rich strand. AID binds specifically to G-loops within transcribed S regions and c-MYC, and G-loops in c-MYC map to the regions associated with translocation breakpoints and aberrant hypermutation in B-cell lymphomas. Aberrant targeting of AID to DNA structures formed upon c-MYC transcription may therefore contribute to the genetic instability of c-MYC in B-cell malignancies.

Animals↗

Specificity of human IgM M-proteins that bind to myelin-associated glycoprotein: peptide mapping, deglycosylation, and competitive binding studies.

Ten patients with neuropathy and IgM M-proteins that bind to the myelin-associated glycoprotein (MAG) were studied to determine whether the M-proteins bind to common regions of MAG and whether the reactive determinants contain carbohydrate residues. The M-protein of one patient was biotinylated, and binding to human MAG was quantitated by enzyme-linked immunosorbent assay (ELISA) by using avidin-biotin-peroxidase complexes. Serum from the same patient and nine others, but not from controls, competed with the biotinylated M-protein for binding to human MAG. Bovine MAG was digested with staph protease or cleaved with cyanogen bromide, and the resultant fragments were separated by electrophoresis and were transferred onto nitrocellulose sheets. Serum from all patients immunostained the peptide fragments identically. Bovine MAG was deglycosylated by trifluoromethanesulfonic acid, and binding of the M-proteins to MAG and to deglycosylated MAG was tested by immunoblotting. None of the patient's M-proteins bound to deglycosylated MAG. Deglycosylated MAG was visualized by using a mouse monoclonal antibody, GEN-S3, directed at the polypeptide core of MAG. The effectiveness of deglycosylation was ascertained by electrophoresis and by binding of biotinylated concanavalin A. These data and the observed identical species specificity of the M-proteins suggest that the respective anti-MAG M-proteins all bind to the same region in MAG and that the reactive determinants may contain carbohydrate moieties.

Animals↗

[Demonstration and possible prognostic value of the nucleolar organizer region (NOR), the microtubule-associated proteins 1A/1B (MAP) and the intermediate filaments vimentin and cytokeratin 19 in invasive ductal breast carcinoma].

We examined the expression of the nucleolar organizer region, the microtubule-associated protein and the intermediate filaments vimentin and cytokeratin 19 and their possible value for prognosis in 51 infiltrating ductal breast carcinomas. We registered the highest NOR counts in pT2/3-tumors, in poorly differentiated breast carcinomas, in tumors with a strong MAP- and vimentin expression (this holds true for vimentin only in pT2/3-tumors). There was a relationship between areas of NOR and the differentiation degree on the one hand and the MAP- and vimentin expression on the other hand. The area of nucleolus increased with the decrease of differentiation and with the increase of vimentin expression, respectively. The expression of cytokeratin 19 showed no homogeneous relations to the traditional prognostic factors as well as to the parameters of the NOR. The area of nucleus had no prognostic value in our study. The value of the NOR is limited by its high variation. The NORs are suitable only as an adjuvant factor for the prognosis. Further studies into MAP and vimentin and their role in the prognosis of infiltrating ductal breast carcinomas appear to be important.

Age Factors↗

Mapmodulin: a possible modulator of the interaction of microtubule-associated proteins with microtubules.

We have purified and characterized a 31-kDa protein named mapmodulin that binds to the microtubule-associated proteins (MAPs) MAP2, MAP4, and tau. Mapmodulin binds free MAPs in strong preference to microtubule-associated MAPs, and appears to do so via the MAP's tubulin-binding domain. Mapmodulin inhibits the initial rate of MAP2 binding to microtubules, a property that may allow mapmodulin to displace MAPs from the path of organelles translocating along microtubules. In support of this possibility, mapmodulin stimulates the microtubule- and dynein-dependent localization of Golgi complexes in semi-intact CHO cells. To our knowledge, mapmodulin represents the first example of a protein that can bind and potentially regulate multiple MAP proteins.

Amino Acid Sequence↗

MAP 4: a microtubule-associated protein specific for a subset of tissue microtubules.

The cytological distribution of microtubule-associated protein 4 (MAP 4) (L. M. Parysek, C. F. Asnes, J. B. Olmsted, 1984, J. Cell Biol., 99:1309-1315) in mouse tissues has been examined. Adjacent 0.5-0.9-micron sections of polyethylene glycol-embedded tissues were incubated with affinity-purified MAP 4 or tubulin antibodies, and the immunofluorescent images were compared. Tubulin antibody labeling showed distinct microtubules in all tissues examined. MAP 4 antibody also labeled microtubule-like patterns, but the extent of MAP 4 reactivity was cell type-specific within each tissue. MAP 4 antibody labeled microtubules in vascular elements of all tissues and in other cells considered to have supportive functions, including Sertoli cells in the testis and glial elements in the nervous system. Microtubule patterns were also observed in cardiac, smooth, and skeletal (eye) muscle, podocytes in kidney, Kuppfer cells in liver, and spermatid manchettes. The only MAP 4-positive cells in which the pattern was not microtubule-like were the principal cells of the collecting ducts in kidney cortex, in which diffuse fluorescence was seen. MAP 4 antibody did not react with microtubule-rich neuronal elements of the central and peripheral nervous system, skeletal muscle from anterior thigh, liver parenchymal cells, columnar epithelial cells of the small intestine, and absorptive cells of the tubular component of the nephron. These observations indicate that MAP 4 may be associated with only certain kinds of cell functions as demonstrated by the preferential distribution with microtubules of defined cell types.

Animals↗

Differentiation of human melanoma cells through p38 MAP kinase is associated with decreased retinoblastoma protein phosphorylation and cell cycle arrest.

The retinoblastoma protein (pRB), the product of the retinoblastoma gene, is a key regulator of the cell cycle, affecting apoptosis, proliferation and differentiation. Dysregulation of pRB is implicated in the pathogenesis of many cancers, including malignant melanoma. Recently we demonstrated that alpha-melanocyte-stimulating hormone (alpha-MSH)-induced activation of p38 mitogen-activated protein (MAP) kinase leads to differentiation of B16 murine melanoma cells. The current study assesses the ability of alpha-MSH to activate p38 MAP kinase in COLO 853 human melanoma cells and determines whether this is linked to modulation of pRB activity. Treatment of COLO 853 cells with alpha-MSH induced time- and concentration-dependent increases in the phosphorylation of p38 MAP kinase, which corresponded with its ability to induce melanogenesis and inhibit cell growth. SB 203580, a selective inhibitor of p38 MAP kinase, blocked both the alpha-MSH-induced melanogenic response and inhibition of cell growth. Cell cycle analysis using flow cytometry revealed that treatment of COLO 853 cells with alpha-MSH for 72 h led to an increase in the proportion of cells in the G(1) phase and a marked reduction in the amount of phosphorylated pRB. Both of these effects were reversed by pre-treatment of cells with SB 203580. In summary, we have demonstrated for the first time that the alpha-MSH-induced differentiation of COLO 853 human melanoma cells proceeds via a p38 MAP kinase-mediated pathway and is associated with decreased pRB phosphorylation and accumulation of cells in the G(1) phase.

Cell Differentiation↗

Fine mapping by genetic association implicates the chromosome 1q23.3 gene UHMK1, encoding a serine/threonine protein kinase, as a novel schizophrenia susceptibility gene.

BACKGROUND: Linkage studies by us and others have confirmed that chromosome 1q23.3 is a susceptibility locus for schizophrenia. Based on this information, several research groups have published evidence that markers within both the RGS4 and CAPON genes, which are 700 kb apart, independently showed allelic association with schizophrenia. Tests of allelic association with both of these genes in our case control sample were negative. Therefore, we carried out further fine mapping between the RGS4 and CAPON genes. METHODS: Twenty-nine SNP and microsatellite markers in the 1q23.3 region were genotyped in the United Kingdom based sample of 450 cases and 450 supernormal control subjects. RESULTS: We detected positive allelic association after the eighth marker was genotyped and found that three microsatellite markers (p = .011, p = .014, p = .049) and two SNPs (p = .004, p = .043) localized in the 700 kb region between the RGS4 and CAPON genes, within the UHMK1 gene, were associated with schizophrenia. Tests of significance for marker rs10494370 remained significant following Bonferroni correction (alpha = .006) for multiple tests. Tests of haplotypic association were also significant for UHMK1 (p = .009) using empirical permutation tests, which make it unnecessary to further correct for both multiple alleles and multiple markers. CONCLUSIONS: These results provide preliminary evidence that the UHMK1 gene increases susceptibility to schizophrenia. Further confirmation in adequately powered samples is needed. UHMK1 is a serine threonine kinase nuclear protein and is highly expressed in regions of the brain implicated in schizophrenia.

Adaptor Proteins, Signal Transducing↗

Regional admixture mapping and structured association testing: conceptual unification and an extensible general linear model.

Individual genetic admixture estimates, determined both across the genome and at specific genomic regions, have been proposed for use in identifying specific genomic regions harboring loci influencing phenotypes in regional admixture mapping (RAM). Estimates of individual ancestry can be used in structured association tests (SAT) to reduce confounding induced by various forms of population substructure. Although presented as two distinct approaches, we provide a conceptual framework in which both RAM and SAT are special cases of a more general linear model. We clarify which variables are sufficient to condition upon in order to prevent spurious associations and also provide a simple closed form "semiparametric" method of evaluating the reliability of individual admixture estimates. An estimate of the reliability of individual admixture estimates is required to make an inherent errors-in-variables problem tractable. Casting RAM and SAT methods as a general linear model offers enormous flexibility enabling application to a rich set of phenotypes, populations, covariates, and situations, including interaction terms and multilocus models. This approach should allow far wider use of RAM and SAT, often using standard software, in addressing admixture as either a confounder of association studies or a tool for finding loci influencing complex phenotypes in species as diverse as plants, humans, and nonhuman animals.

Computer Simulation↗

In vivo association between p56lck and MAP kinase during IL-2-mediated lymphocyte proliferation.

We previously reported that p56lck expression is upregulated in human B lymphocytes upon mitogenic stimulation. In this report, we characterized the molecules associated with p56lck in vivo in leukemic B cells costimulated with anti-mu Ab and IL-2 for 72 h. In vitro phosphorylation after p56lck immunoprecipitation indicated that p56lck is associated in vivo with the beta chain of the IL-2 receptor and p42 MAP kinase as well as a number of other proteins. Moreover, p56lck-associated MAP kinase is tyrosine and threonine phosphorylated, suggesting that it is activated. Prevention of DNA synthesis with aphidicolin abrogated this molecular association, and furthermore, cell cycle analysis with IL-2-dependent T cells showed that in cells in G1, MAP kinase was not associated to p56lck, whereas this p56lck-MAP kinase association was observed when cells are in S phase. Thus, p56lck and MAP kinase are only associated during S phase. These data suggest that MAP kinase in association with p56lck is directly involved in the control of IL-2-mediated DNA synthesis of both B and T lymphocytes.

Aphidicolin↗

Gene mapping by linkage and association analysis.

Genetic analysis is used to map genes, including disease loci, to positions within the human genome. Linkage analysis depends on the co-segregation of a gene (locus) and a phenotype through a pedigree, while association analysis, or linkage disequilibrium mapping, depends on measuring deviation from the random occurrence of alleles in a haplotype in unrelated individuals or nuclear families. Complex computer programs may be used in both forms of analysis. In recent years most interest has focused on identifying genes involved in common, multifactorial diseases. Here I review some current and developing techniques of genetic analysis and give references to where further information can be obtained.

Animals↗

A disease-associated germline deletion maps the type 2 neurofibromatosis (NF2) gene between the Ewing sarcoma region and the leukaemia inhibitory factor locus.

RFLP typing of members of a neurofibromatosis type 2 (NF2) family suggested that affected individuals were hemizygous at the neurofilament heavy chain (NEFH) locus, possibly as a result of a disease-associated deletion. Conventional karyotyping revealed no evidence for a deletion and all or a majority of the affected family members were heterozygous for closely linked markers which mapped proximal to the NEFH locus (D22S1 and D22S56) and for the distal marker D22S32. FISH analysis confirmed a disease-associated germinal deletion on 22q which encompassed the NEFH locus, which is known to be very closely linked to NF2, but did not extend as far as the proximal Ewing sarcoma region or the distal leukaemia factor (LIF) locus. PFGE analysis with a LIF cosmid subclone identified patient-specific NotI and MluI fragments and suggested that the deletion is about 700 kb in length. Although this large deletion could be expected to eliminate a considerable fraction, and possibly all of the NF2 gene, the resulting phenotype is the mild, so-called Gardner subtype of NF2. The deletion should provide a useful mapping resource for characterising the chromosomal region containing the NF2 locus.

Alleles↗

Analysis of the HLA-ABC linkage disequilibrium: decreasing strength of gametic association with increasing map distance.

1242 HLA-ABC haplotypes of the North German population (Hambrug) as deduced by family analyses are described. They are in perfect agreement with recently published data by Mayr (1977) from Austria (Vienna) in all parameters tested: frequency of the single HLA-alleles, haplotype distribution and linkage disequilibrium values. Gametic association studies revealed that 69.4% of the B and C genes (map distance 0.2cM) 36.9% of the A and C genes (0.6 cM), but only 23.2% of the A and B genes (0.8 cM) were significantly more often combined than expected due to their frequencies. From these findings it seems likely that the linkage disequilibrium within the MHC is rather due to a short evolutionary period than to selective forces. Some observations as to the most common European haplotype A1,B8 are discussed.

Alleles↗

Mapping the HLA association in Behçet's disease: a role for tumor necrosis factor polymorphisms?

OBJECTIVE: Experimental evidence suggests that inappropriate regulation of tumor necrosis factor alpha (TNF alpha) may play a role in the pathogenesis of Behçet's disease (BD). This is supported by recent reports highlighting the efficacy of anti-TNF alpha agents in the treatment of this disease. The TNF gene is encoded in the class III region of the HLA complex adjacent to HLA-B. This genetic proximity to a gene that is already widely implicated in disease susceptibility led us to investigate the association between TNF promoter polymorphisms and susceptibility to BD. METHODS: We studied 133 UK white Caucasoid patients with BD and 354 healthy controls. We attempted to dissect the contribution of individual polymorphisms in this gene-dense region by linkage disequilibrium mapping across 6 adjacent genes. RESULTS: We report a novel association with the TNF promoter allele TNF-1031C. Subsequent analysis identified 2 extended HLA haplotypes associated with BD. One of them contained the previously recognized susceptibility gene HLA-B*51, while the other was defined by HLA-B*5701. Both of these haplotypes contained the TNF promoter polymorphism -1031C, an allele that was associated with disease even in individuals who did not carry either HLA-B*51 or HLA-B*5701. CONCLUSION: The TNF-1031C allele is independently associated with susceptibility to BD in Caucasoid patients. Further studies will be required to determine the functional effects of this polymorphism, its influence in disease pathogenesis, and its role in other ethnic groups.

Behcet Syndrome↗

QTL mapping of ten agronomic traits on the soybean ( Glycine max L. Merr.) genetic map and their association with EST markers.

A set of 184 recombinant inbred lines (RILs) derived from soybean vars. Kefeng No.1 x Nannong 1138-2 was used to construct a genetic linkage map. The two parents exhibit contrasting characteristics for most of the traits that were mapped. Using restricted fragment length polymorphisms (RFLPs), simple sequence repeats (SSRs) and expressed sequence tags (ESTs), we mapped 452 markers onto 21 linkage groups and covered 3595.9 cM of the soybean genome. All of the linkage groups except linkage group F were consistent with those of the consensus map of Cregan et al. (1999). Linkage group F was divided into two linkage groups, F1 and F2. The map consisted of 189 RFLPs, 219 SSRs, 40 ESTs, three R gene loci and one phenotype marker. Ten agronomic traits-days to flowering, days to maturity, plant height, number of nodes on main stem, lodging, number of pods per node, protein content, oil content, 100-seed weight, and plot yield-were studied. Using winqtlcart, we detected 63 quantitative trait loci (QTLs) that had LOD>3 for nine of the agronomic traits (only exception being seed oil content) and mapped these on 12 linkage groups. Most of the QTLs were clustered, especially on groups B1 and C2. Some QTLs were mapped to the same loci. This pleiotropism was common for most of the QTLs, and one QTL could influence at most five traits. Seven EST markers were found to be linked closely with or located at the same loci as the QTLs. EST marker GmKF059a, encoding a repressor protein and mapped on group C2, accounted for about 20% of the total variation of days to flowering, plant height, lodging and nodes on the main stem, respectively.

Agriculture↗

Bayesian association-based fine mapping in small chromosomal segments.

A Bayesian method for fine mapping is presented, which deals with multiallelic markers (with two or more alleles), unknown phase, missing data, multiple causal variants, and both continuous and binary phenotypes. We consider small chromosomal segments spanned by a dense set of closely linked markers and putative genes only at marker points. In the phenotypic model, locus-specific indicator variables are used to control inclusion in or exclusion from marker contributions. To account for covariance between consecutive loci and to control fluctuations in association signals along a candidate region we introduce a joint prior for the indicators that depends on genetic or physical map distances. The potential of the method, including posterior estimation of trait-associated loci, their effects, linkage disequilibrium pattern due to close linkage of loci, and the age of a causal variant (time to most recent common ancestor), is illustrated with the well-known cystic fibrosis and Friedreich ataxia data sets by assuming that haplotypes were not available. In addition, simulation analysis with large genetic distances is shown. Estimation of model parameters is based on Markov chain Monte Carlo (MCMC) sampling and is implemented using WinBUGS. The model specification code is freely available for research purposes from http://www.rni.helsinki.fi/~mjs/.

Algorithms↗