Multiple Tandem Repeats Research Articles

Decreasing amplification bias associated with multiple displacement amplification and short tandem repeat genotyping

Although multiple displacement amplification (MDA) is being used increasingly to amplify genomes, the amplification bias generated by the ϕ29 polymerase can be a concern with genotyping applications. It has been noted that the bias is pronounced with small template amounts, particularly with single nucleotide polymorphism (SNP) and short tandem repeat (STR) genotyping. Bias may occur between loci, or between alleles within a locus, and may differ between sample donors at the same loci. Previous research has suggested that omitting denaturation of the template prior to amplification can reduce the observed bias significantly. Comparison of the two methods (with and without denaturation) has found that nondenaturation of template reverses the direction of bias observed between allelic pairs following MDA. By combining two MDA reactions, one denatured and one nondenatured, the bias was found to be reduced significantly, aiding copy number analysis and subsequent genotyping.

N.BspD6I DNA nickase strongly stimulates template-independent synthesis of non-palindromic repetitive DNA by Bst DNA polymerase

Highly efficient DNA synthesis without template and primer DNAs occurs when N.BspD6I DNA nickase is added to a reaction mixture containing deoxynucleoside triphosphates and the large fragment of Bst DNA polymerase. Over a period of 2 h, virtually all the deoxynucleoside triphosphates (dNTPs) become incorporated into DNA. Inactivation of N.BspD6I nickase by heating inhibits DNA synthesis. Optimal N.BspD6I activity is required to achieve high yields of synthesized DNA. Electron microscopy data revealed that the majority of DNA molecules have a branched structure. Cloning and sequencing of the fragments synthesized demonstrated that the DNA product mainly consists of multiple hexanucleotide non-palindromic tandem repeats containing nickase recognition sites. A possible mechanism is discussed that addresses template-independent DNA synthesis stimulated by N.BspD6I nickase.

S-layer-mediated display of the immunoglobulin G-binding domain of streptococcal protein G on the surface of Caulobacter crescentus: development of an immunoactive reagent.

The immunoglobulin G (IgG)-binding streptococcal protein G is often used for immunoprecipitation or immunoadsorption-based assays, as it exhibits binding to a broader spectrum of host species IgG and IgG subclasses than the alternative, Staphylococcus aureus protein A. Caulobacter crescentus produces a hexagonally arranged paracrystalline protein surface layer (S-layer) composed of a single secreted protein, RsaA, that is notably tolerant of heterologous peptide insertions while maintaining the surface-attached crystalline character. Here, a protein G IgG-binding domain, GB1, was expressed as an insertion into full-length RsaA on the cell surface to produce densely packed immunoreactive particles. GB1 insertions at five separate sites were expressed, and all bound rabbit and goat IgG, but expression levels were reduced compared to those of wild-type RsaA and poor binding to mouse IgG was noted. To remedy this, we used the 20-amino-acid Muc1 peptide derived from human mucins as a spacer, since insertions of multiple tandem repeats were well tolerated for RsaA secretion and assembly. This strategy worked remarkably well, and recombinant RsaA proteins, containing up to three GB1 domains, surrounded by Muc1 peptides, not only were secreted and assembled but did so at wild-type levels. The ability to bind IgG (including mouse IgG) increased as GB1 units were added, and those with three GB1 domains bound twice as much rabbit IgG per cell as S. aureus cells (Pansorbin). The ability of recombinant protein G-Caulobacter cells to function as immunoactive reagents was assessed in an immunoprecipitation assay using a FLAG-tagged protein and anti-FLAG mouse monoclonal antibody; their performance was comparable to that of protein G-Sepharose beads. This work demonstrates the potential for using cells expressing recombinant RsaA/GB1 in immunoassays, especially considering that protein G-Caulobacter cells are more cost-effective than protein G beads and exhibit a broader species and IgG isotype binding range than protein A.

Application of variable number of tandem repeat analysis to determine the origin of S. aureus contamination from milk to cheese in goat cheese farms

The aim of the paper was to evaluate multiple locus variable number tandem repeat analysis and staphylokinase gene amplification to track the origins of Staphylococcus aureus contamination in dairy environments. The diversity of MLVA profiles (58 profiles among 481 strains) allowed identification of several sources of cheese contamination, most often from bulk milk through S. aureus-shedding goat milk and occasionally milking machine or human. The staphylokinase gene was always present in human S. aureus strains, although it was also present in those from S. aureus-shedding goat milks and, to a lesser degree, from bulk milk and cheeses. Contamination may persist over the same year and also over 2 consecutive years.

Cellular and molecular studies of Marfan syndrome mutations identify co-operative protein folding in the cbEGF12–13 region of fibrillin-1

Human fibrillin-1 is an extra-cellular matrix glycoprotein with a modular organisation that includes 43 calcium-binding epidermal growth factor-like (cbEGF) domains arranged as multiple tandem repeats interspersed with transforming growth factor beta binding protein-like (TB) domains. We have studied Marfan syndrome-causing mutations which affect calcium binding to cbEGF13, and demonstrate that in human fibroblast cells they cause unexpected endoplasmic reticulum retention, indicative of a folding defect. Biochemical and biophysical studies of in vitro refolded fragments from the TB3-cbEGF14 region indicate long-range and unidirectional effects of these substitutions on the adjacent N-terminal domain cbEGF12. In contrast, only short-range effects of a pathogenic mutation affecting calcium binding to cbEGF19 are observed, and secretion of this mutant protein occurs. Further NMR studies on wild-type cbEGF12-13 and cbEGF12-14 identify a co-operative dependence of domain folding where calcium binding to cbEGF13 is required before cbEGF12 can adopt a native Ca(2+)-dependent fold. These data demonstrate that during biosynthesis of fibrillin-1, multiple tandem repeats of cbEGF domains may not necessarily fold independently and therefore missense mutations resulting in identical substitutions may have different effects on the fate of the mutant protein. Complex folding of modular proteins should therefore be considered when interpreting the molecular pathology of single-gene disorders.

Cloning, sequencing and molecular characterisation of a cryptic plasmid from a urease-positive thermophilic Campylobacter (UPTC) isolate

Cloning, sequencing and molecular characterisation of a cryptic plasmid, pUPTC237, from a urease-positive thermophilic Campylobacter (UPTC) isolate obtained from the natural environment in Northern Ireland is reported in this study. Based on the determined DNA sequence, the pUPTC237 DNA was identified as a circular molecule of 3828 bp with a G+C content of 29.5%. As with other plasmid DNAs from Gram-negative bacteria, pUPTC237 contained an A+T-rich region (A+T content: 95%), followed by multiple direct tandem repeat units of 22 bp, characteristic of a replication origin and iteron sequence. A possible open reading frame (ORF)-1 was located upstream of the A+T-rich region and the iteron sequence that encoded a 460 amino acid protein similar to the mobilisation (mob) protein and two putative promoter structure sequences at the -35 and -10 regions and a possible ribosome binding site occurred upstream of the start codon for the ORF-1. Moreover, three possible ORFs (a short ORF-2 encoding 26 amino acids, similar to repA; an ORF-3 encoding 341 amino acids, similar to repB; and an ORF-4 encoding 96 amino acids with unknown function) were also identified. There are also two putative promoter structures for these three ORFs at the -35 and -10 regions upstream of the possible ORF-2. A possible transcription termination region was identified downstream of ORF-4. Northern blot hybridisation analysis suggested that these four ORFs constitute an operon and generate a messenger RNA (mRNA) transcript.

Molecular crowding increases the amplification success of multiple displacement amplification and short tandem repeat genotyping

Multiple displacement amplification (MDA) can generate large quantities of genomic DNA product from small amounts of template. We investigated the ability of MDA to amplify samples containing very small amounts of target DNA (5 pg to 1 ng) in the presence of a second, larger DNA sample for downstream short tandem repeat (STR) multiplex genotyping. We observed that STR amplification success of the minor fraction was increased in these mixed samples when compared with standard PCR only or MDA containing only the single trace DNA sample. Increased numbers of alleles were detected, with less amplification bias between loci than in single source samples undergoing the same protocol. To improve the STR genotyping accuracy, animal DNA was substituted for the additional human DNA, maintaining the increase in the number and quality of human-specific STR loci amplified. Polyethylene glycol 400, a commonly used crowding agent, was used as a replacement for the added genomic DNA in the MDA reaction and produced very similar results. Therefore, we suggest that additional DNA is acting as a molecular crowding agent during MDA. Performing MDA on trace amounts of DNA under crowded conditions results in greater numbers of alleles being amplified and more balanced amplification occurring between alleles.

Ten tandem repeats of β-hCG 109–118 enhance immunogenicity and anti-tumor effects of β-hCG C-terminal peptide carried by mycobacterial heat-shock protein HSP65

The β-subunit of human chorionic gonadotropin (β-hCG) is secreted by many kinds of tumors and it has been used as an ideal target antigen to develop vaccines against tumors. In view of the low immunogenicity of this self-peptide,we designed a method based on isocaudamer technique to repeat tandemly the 10-residue sequence X of β-hCG (109–118), then 10 tandemly repeated copies of the 10-residue sequence combined with β-hCG C-terminal 37 peptides were fused to mycobacterial heat-shock protein 65 to construct a fusion protein HSP65-X10-βhCGCTP37 as an immunogen. In this study, we examined the effect of the tandem repeats of this 10-residue sequence in eliciting an immune by comparing the immunogenicity and anti-tumor effects of the two immunogens, HSP65-X10-βhCGCTP37 and HSP65-βhCGCTP37 (without the 10 tandem repeats). Immunization of mice with the fusion protein HSP65-X10-βhCGCTP37 elicited much higher levels of specific anti-β-hCG antibodies and more effectively inhibited the growth of Lewis lung carcinoma (LLC) in vivo than with HSP65-βhCGCTP37, which should suggest that HSP65-X10-βhCGCTP37 may be an effective protein vaccine for the treatment of β-hCG-dependent tumors and multiple tandem repeats of a certain epitope are an efficient method to overcome the low immunogenicity of self-peptide antigens.

Genotyping of Bacillus anthracis strains based on automated capillary 25-loci Multiple Locus Variable-Number Tandem Repeats Analysis

BackgroundThe genome of Bacillus anthracis, the etiological agent of anthrax, is highly monomorphic which makes differentiation between strains difficult. A Multiple Locus Variable-number tandem repeats (VNTR) Analysis (MLVA) assay based on 20 markers was previously described. It has considerable discrimination power, reproducibility, and low cost, especially since the markers proposed can be typed by agarose-gel electrophoresis. However in an emergency situation, faster genotyping and access to representative databases is necessary.ResultsGenotyping of B. anthracis reference strains and isolates from France and Italy was done using a 25 loci MLVA assay combining 21 previously described loci and 4 new ones. DNA was amplified in 4 multiplex PCR reactions and the length of the resulting 25 amplicons was estimated by automated capillary electrophoresis. The results were reproducible and the data were consistent with other gel based methods once differences in mobility patterns were taken into account. Some alleles previously unresolved by agarose gel electrophoresis could be resolved by capillary electrophoresis, thus further increasing the assay resolution. One particular locus, Bams30, is the result of a recombination between a 27 bp tandem repeat and a 9 bp tandem repeat. The analysis of the array illustrates the evolution process of tandem repeats.ConclusionIn a crisis situation of suspected bioterrorism, standardization, speed and accuracy, together with the availability of reference typing data are important issues, as illustrated by the 2001 anthrax letters event. In this report we describe an upgrade of the previously published MLVA method for genotyping of B. anthracis and apply the method to the typing of French and Italian B. anthracis strain collections. The increased number of markers studied compared to reports using only 8 loci greatly improves the discrimination power of the technique. An Italian strain belonging to the B branch was described, and two new branches, D and E, are proposed. Owing to the upgrading achieved here, precise genotyping can now be produced either by automated capillary electrophoresis, or by the more accessible but slower and for some markers slightly less accurate agarose gel methodology.

Establishment of a genotyping scheme forCoxiella burnetii

Coxiella burnetii is the causative agent of Q fever. The bacterium is highly infectious and is classified as a category B biological weapon. The tools of molecular biology are of utmost importance in a rapid and unambiguous identification of C. burnetii in naturally occurring Q fever outbreaks, or in cases of a deliberate release of the infectious agent. In this work, development of a multiple locus variable number tandem repeats (VNTR) analysis (MLVA) for the characterization of C. burnetii is described. Sixteen C. burnetii isolates and five passage history/laboratory variants were characterized. The VNTR markers revealed many polymorphisms resulting in nine unique MLVA types that cluster into five different clusters. This proves that the MLVA system is highly discriminatory. The selected VNTR markers were stable. The MLVA method developed in this report is a promising tool for the characterization of C. burnetii isolates and their epidemiological study.

Structural basis for phosphorylation-dependent signaling in the DNA-damage response

The response of eukaryotic cells to DNA damage requires a multitude of protein-protein interactions that mediate the ordered repair of the damage and the arrest of the cell cycle until repair is complete. Two conserved protein modules, BRCT and forkhead-associated (FHA) domains, play key roles in the DNA-damage response as recognition elements for nuclear Ser/Thr phosphorylation induced by DNA-damage-responsive kinases. BRCT domains, first identified at the C-terminus of BRCA1, often occur as multiple tandem repeats of individual BRCT modules. Our recent structural and functional work has revealed how BRCT repeats recognize phosphoserine protein targets. It has also revealed a secondary binding pocket at the interface between tandem repeats, which recognizes the amino-acid 3 residues C-terminal to the phosphoserine. We have also studied the molecular function of the FHA domain of the DNA repair enzyme, polynucleotide kinase (PNK). This domain interacts with threonine-phosphorylated XRCC1 and XRCC4, proteins responsible for the recruitment of PNK to sites of DNA-strand-break repair. Our studies have revealed a flexible mode of recognition that allows PNK to interact with numerous negatively charged substrates.

Comparative Study of Chimerism Dynamics between Allogenic Bone Marrow Transplantation and Nonmyeloablative Stem Cell Transplantation.

Objective To explore whether the dynamics and outcome of chimerism are different between allogenic bone marrow transplantation (allo-BMT) and nonmyeloablative stem cell transplantation (NST) these two kinds of transplant strategies in order to define factors that significantly influence the early engraftment of donor stem cells.Methods A retrospective comparative study was performed between 20 patients undergoing BMT and 18 patients receiving NST. There are several differences between NST and standard allo-BMT, such as conditioning regimens, the source and the quantity of hematopoietic stem cells and the protocal of GVHD prophylaxis. But component of two groups patients were similar. Sequential monitoring of chimerism were performed in 38 patients in different periods post transplantation using multiple short tandem repeat(STR) amplification by fluorescence labeling polymerase chain reaction(PCR) combined with a capillary electrophoresis.ResultsThe median age of recipient, median quantity of MNC, CD34+ cells and T lymphocyte cells of NST were significantly higher compared with the BMT. The median time for absolute neutrophils recovery of NST(without G-CSF) was similar to that of BMT. While compared to BMT, NST lead to faster engraftment of platelets.The establishment of full donor chimerism (FDC) of NST was much more earlier than that of BMT (1 month VS 3 months, p=0.01). In the early post-transplant period(up to day 30), there was an apparent increase in the incidence of full donor chimerism (FDC) following NST compared with BMT(38.9% VS 20%, p=0.019). Whereas a significantly lower incidence of mixed chimerism(MC) occurred in recipients of NST compare to BMT.(61.2% VS 80%, p=0.02). It suggest that in the early stage after transplantation, the engraftment of donor cells of NST was more rapid than that of BMT. The major relevant factor causing full donor chimerism formation seems to be the high quantity of donor CD34+ cells and T lymphocyte cells.Fludarabine-based reduced-intensity conditioning didn't result in a significant delay of donor cell engraftment compared to standard conditioning regimen.The incidence of chronic GVHD was much higher in group of NST than that of BMT group(80% VS 50%, p=0.007).This finding was related to that patients of NST group received the higher doses of CD34+ cells. Conclusion Factors that significantly influence the early kinetics of donor chimerism and donor cell engraftment after transplantation were the quantity of stem cell and T lymphocyte cell of grafts.

Preimplantation Genetics: Improving Access to Stem Cell Therapy

There has been progress in the application of stem cell transplantation for treatment of an increasing number of severe congenital and acquired bone marrow disorders, currently restricted by the availability of human leukocyte antigen (HLA)-matched related donors. Preimplantation HLA typing has recently been introduced to improve the access to stem cell therapy for inherited bone marrow failures. Preimplantation genetic diagnosis (PGD) provides an option not only for avoiding an affected pregnancy with thalassemia and other inherited disorders but also for preselection of the HLA-compatible donors for affected siblings. Multiple short tandem repeat markers throughout the HLA region are applied for this purpose, allowing 100% accuracy of HLA typing, through picking up possible recombination in the HLA region, as well as the copy number of chromosome 6, which affect accuracy of preimplantation HLA typing. Present experience of preimplantation HLA typing includes preimplantation HLA typing in 180 cycles, 122 of which were done as part of PGD for Fanconi anemia, thalassemia, Wiscott-Aldrich syndrome, hyper-immunoglobulin M syndrome, hypohidrotic ectodermal dysplasia with immune deficiency, and X-linked adrenoleukodystrophy, and 58 for the sole purpose of HLA typing for leukemias and for aplastic and Diamond-Blackfan anemia. The applied method resulted in the accurate preselection and transfer of 100% HLA-matched embryos, yielding already three dozen clinical pregnancies and the birth of two dozen HLA-matched children to the siblings requiring stem cell transplantation. Successful therapy with HLA-matched stem cells, obtained from these PGD children, has been achieved already for Diamond-Blackfan anemia hypohidrotic ectodermal dysplasia with immune deficiency and thalassemia.

Bone Marrow Engraftment Analysis after Granulocyte Transfusion

We present the case of a 6-year-old male who received an allogeneic bone marrow transplant as part of treatment for acute lymphoblastic leukemia. The patient relapsed 5 months after transplantation and received additional chemotherapy. He acquired an angioinvasive fungal infection that required transfusion of granulocytes. Approximately 5 weeks after relapsing (181 days after transplant), a bone marrow specimen was taken for molecular engraftment analysis and flow cytometry to assess graft loss as well as residual disease. The engraftment results generated by the multiple short tandem repeat loci tested were inconsistent, and alleles were present at several loci that were of neither patient nor donor origin. An error in specimen identification was initially considered. Further investigation into the circumstances surrounding procurement of the patient's bone marrow aspirate revealed that the patient had received a granulocyte transfusion approximately 10 hours before the bone marrow specimen was taken. In addition, morphological and flow cytometric analyses of the same bone marrow aspirate demonstrated a significant degree of peripheral blood contamination. We determined that the unknown alleles in the bone marrow engraftment specimen were derived from the donor of the transfused granulocytes. This case illustrates that white cell transfusion can lead to erroneous bone marrow engraftment results, particularly if only one microsatellite locus is used to monitor engraftment.

Analysis of the role of HP0208, a phase-variable open reading frame, and its homologues HP1416 and HP0159 in the biosynthesis of Helicobacter pylori lipopolysaccharide

The roles of the three ORFs HP0208, HP0159 and HP1416 in the biosynthesis of Helicobacter pylori 26695 LPS were investigated in this study. These ORFs represent a paralogous family of genes with homology to the Salmonella enterica serovar Typhimurium (hereafter referred to as S. typhimurium) waaJ gene, which encodes an alpha-1,2-glycosyltransferase required for core LPS biosynthesis. HP0208 contains multiple tandem repeats of the dimer 5'GA at its 5' end and its expression is predicted to be subject to phase variation. The number of 5'GA repeats present in this ORF was found to be non-permissive for the expression of HP0208 in the majority of H. pylori strains examined. To determine a role for this ORF in LPS biosynthesis a non-phase-variable, constitutively expressed variant of HP0208 was constructed and introduced into the genome of H. pylori 26695. Analysis of the LPS profile of this strain by Tricine-SDS-PAGE and immunoblotting with anti-Lewis Y antigen (Le(y)) mAbs confirmed a role for HP0208 in the biosynthesis of core LPS. A role for HP0159 and HP1416 in the biosynthesis of core LPS was also established. Although homologous to waaJ, H. pylori HP0208, HP0159 and HP1416 failed to complement an S. typhimurium waaJ mutant, suggesting that these ORFs encode functionally different enzymes.

The ExsA Protein of Bacillus cereus Is Required for Assembly of Coat and Exosporium onto the Spore Surface

The outermost layer of spores of the Bacillus cereus family is a loose structure known as the exosporium. Spores of a library of Tn917-LTV1 transposon insertion mutants of B. cereus ATCC 10876 were partitioned into hexadecane; a less hydrophobic mutant that was isolated contained an insertion in the exsA promoter region. ExsA is the equivalent of SafA (YrbA) of Bacillus subtilis, which is also implicated in spore coat assembly; the gene organizations around both are identical, and both proteins contain a very conserved N-terminal cortex-binding domain of ca. 50 residues, although the rest of the sequence is much less conserved. In particular, unlike SafA, the ExsA protein contains multiple tandem oligopeptide repeats and is therefore likely to have an extended structure. The exsA gene is expressed in the mother cell during sporulation. Spores of an exsA mutant are extremely permeable to lysozyme and are blocked in late stages of germination, which require coat-associated functions. Two mutants expressing differently truncated versions of ExsA were constructed, and they showed the same gross defects in the attachment of exosporium and spore coat layers. The protein profile of the residual exosporium harvested from spores of the three mutants--two expressing truncated proteins and the mutant with the original transposon insertion in the promoter region--showed some differences from the wild type and from each other, but the major exosporium glycoproteins were retained. The exsA gene is extremely important for the normal assembly and anchoring of both the spore coat and exosporium layers in spores of B. cereus.

Characterization of genotype–phenotype relationships and stratification by the CARD15 variant genotype for inflammatory bowel disease susceptibility loci using multiple short tandem repeat genetic markers

The classification of ulcerative colitis (UC), Crohn disease (CD), and indeterminate colitis (IC) as forms of inflammatory bowel disease (IBD) is based on clinical, radiological, and histological criteria. The genetic basis of IBD is well founded, and susceptibility loci have been identified on several different chromosomes. We aimed to define genotype-phenotype relationships and interactions with the IBD susceptibility gene CARD15for various IBD susceptibility loci (IBD1, IBD2, IBD5, IBD6, IBD7, and chromosome 4) by characterizing previously described peak LOD score short tandem repeat (STR) markers. The study population consisted of 484 severely affected Caucasian patients with IBD, 144 healthy controls, and 348 nonaffected first-degree relatives of IBD patients. Associations were defined with the use of population- and family-based methodology. Correction for multiple testing was performed with a method based on an experimental false discovery rate. We provide novel evidence to show that IBD2 is involved in susceptibility to IC and terminal ileal CD in this population, with overrepresentation of IBD2 STR D12S83 (GenBank Z16592.1) allele 7 (g.49_60del[CA](6)) in IC (q = 0.038, P = 0.014) and underrepresentation of allele 8 (g.51_60del[CA](5)) in terminal ileal CD (q = 0.038, P = 0.016). The association of IBD2 with IC was confirmed by family-based testing. We also provide novel evidence to show that IBD5 is involved in susceptibility to IC and colonic/ileocolonic CD in this population, with overrepresentation of IBD5 STR D5S1984 (GenBank Z52623.1) allele 5 (g.183_186del[CA](2)) in both IC (q = 0.040, P = 0.005) and colonic/ileocolonic CD (q = 0.040, P = 0.004). Evidence is also given for potential interactions between CARD15and IBD2/IBD5. Other findings include an association of IBD2 with UC, and an association of IBD1 with terminal ileal and colonic/ileocolonic CD.

STRUCTURAL CHARACTERIZATION OF THE TERMINAL DOMAINS OF LINEAR PLASMID‐LIKE DNA FROM THE GREEN ALGA ERNODESMIS (CHLOROPHYTA)1

Terminal regions of linear plasmid‐like DNA molecules from chloroplasts of the green alga Ernodesmis verticillata (Kützing) Børgesen were cloned and structurally characterized. Phosphorylation experiments with polynucleotide kinase indicated the presence of a 5′‐phosphate, but the data did not reveal any protective protein(s) at the 5′ end. To characterize the 3′ end of these molecules, homopolymer‐ (poly(G)‐) tailed molecules were annealed to and cloned into a linearized vector that was poly(C) tailed with terminal deoxynucleotidyl transferase. Sequencing analyses verified the heterogeneous nature of the molecules. Two distinct clones displayed extensive terminal inverted repeats (TIRs) at the 3′ end (94 and 433 nt). Shorter TIRs (3–6 nt) were identified at the 3′ end of most clones, which may serve to protect the ends. In fact, exonuclease III and λ exonuclease digested the plasmid‐like DNAs only after heat denaturation, signifying that conformational changes due to such treatment potentially make the 3′ and 5′ ends (respectively) susceptible to degradation. Multiple tandem and direct repeats were evident near the 3′ ends. A consensus sequence of 18+ nt was discovered in nearly every clone opposite the poly(G) tail, suggesting that this sequence has structural and/or functional significance. Pair‐wise sequence comparisons and the presence of repeats indicated that these novel molecules may be highly recombinant.

Binding patterns of DTR-specific antibodies reveal a glycosylation-conditioned tumor-specific epitope of the epithelial mucin (MUC1).

Glycosylation determines essential biological functions of epithelial mucins in health and disease. We report on the influence of glycosylation of the immunodominant DTR motif of MUC1 on its antigenicity. Sets of novel glycopeptides were synthesized that enabled us to examine sole and combined effects of peptide length (number of repeats) and O-glycosylation with GalNAc at the DTR motif on the binding patterns of 22 monoclonal antibodies recognizing this motif. In case of unglycosylated peptides almost all antibodies bound better to multiple MUC1 tandem repeats. Glycosylation at the DTR led to enhanced binding in 11 cases, whereas 10 antibodies were not influenced in binding, and one was inhibited. In nine of the former cases both length and DTR glycosylation were additive in their influence on antibody binding, suggesting that both effects are different. Improved binding to the glycosylated DTR motif was exclusively found with antibodies generated against tumor-derived MUC1. Based on these data a tumor-specific MUC1 epitope is defined comprising the ...PDTRP... sequence in a particular conformation essentially determined by O-glycosylation at its threonine with either GalNAcalpha1 or a related short glycan. The results can find application in the field of MUC1-based immunotherapy.

90 Initial characterization of the terminal domains of linear plasmid‐like dna's from ernodesmis verticillata (chlorophyta)

Terminal regions of linear plasmid‐like DNAs from the chloroplasts of Ernodesmis verticillata (Kützing) Bøergesen were characterized. The 2.2‐kb molecules are believed to consist of a single strand of DNA folded back on itself in a hairpin configuration. The 5′ end of isolated molecules is capable of being phosphorylated with polynucleotide kinase, and the 3′ end can be homopolymeric tailed with terminal deoxynucleotidyl transferase. Thus, both ends are accessible for further manipulation. Exonuclease III and lambda exonuclease digest native molecules only after heat denaturation and certain cooling regimes, suggesting that conformational changes brought about by de‐ and renaturation make the 3′and 5′ ends (respectively) vulnerable to exonuclease activity. The cloning strategy involved poly‐G tailing of native molecules, and hybridizing them to a poly‐C‐tailed vector. Two rounds of cloning the 3′ ends yielded a total of 14 clones. Molecular sequencing revealed that two distinct clones have extensive terminal inverted repeats (TIRs). Short TIRs are also evident in most clones, possibly indicating that molecular foldbacks protect the 3′ ends. Multiple tandem and direct repeats are also prevalent near the 3′ termini. Despite overall sequence heterogeneity among most clones, a consensus sequence found at the 5′ end of these clones probably has structural and/or functional significance. Pairwise sequence comparisons and the presence of extensive repeats suggest that these molecules may be highly recombinant.