Alkylation of a human telomere sequence by heterotrimeric chlorambucil PI polyamide conjugates

We designed and synthesized human telomere alkylating N-methylpyrrole-N-methylimidazole (PI) polyamide conjugates (1–6). The C-type conjugates 1–3 possessed a chlorambucil moiety at the C terminus, whereas the N-type conjugates 4–6 had one of these moieties at the N terminus. The DNA alkylating activity of these conjugates was evaluated by high-resolution denaturing polyacrylamide gel electrophoresis using a 220 bp DNA fragment containing the human telomere repeat sequence 5′-(GGGTTA)₄-3′/5′-(TAACCC)₄-3′. C-type conjugates are designed to alkylate the G-rich-strand-containing 5′-GGGTTA-3′ and N-type conjugates were designed to alkylate the complementary C-rich strand-containing 5′-TAACCC-3′ sequence. The difference between conjugates 1–3 and 4–6 lies in the linker region between the polyamide moiety and chlorambucil. Conjugates 1 and 4 efficiently alkylated the 5′-GGTTAGGGTTA-3′ and 5′-CCCTAACCCTAA-3′ sequences, respectively, by recognizing 11 bp in the presence of distamycin A (Dist), in a heterotrimeric manner: one long alkylating polyamide conjugate (1–6) and two short partners (Dist).     

Combinatorial recognition of a complex telomere repeat sequence by the Candida parapsilosis Cdc13AB heterodimer

The telomere repeat units of Candida species are substantially longer and more complex than those in other organisms, raising interesting questions concerning the recognition mechanisms of telomere-binding proteins. Herein we characterized the properties of Candida parapsilosis Cdc13A and Cdc13B, two paralogs that are responsible for binding and protecting the telomere G-strand tails. We found that Cdc13A and Cdc13B can each form complexes with itself and a heterodimeric complex with each other. However, only the heterodimer exhibits high-affinity and sequence-specific binding to the telomere G-tail. EMSA and crosslinking analysis revealed a combinatorial mechanism of DNA recognition, which entails the A and B subunit making contacts to the 3′ and 5′ region of the repeat unit. While both the DBD and OB4 domain of Cdc13A can bind to the equivalent domain in Cdc13B, only the OB4 complex behaves as a stable heterodimer. The unstable Cdc13AB_DBD complex binds G-strand with greatly reduced affinity but the same sequence specificity. Thus the OB4 domains evidently contribute to binding by promoting dimerization of the DBDs. Our investigation reveals a rare example of combinatorial recognition of single-stranded DNA and offers insights into the co-evolution of telomere DNA and cognate binding proteins.     

Acquisition of telomere repeat sequences by transfected DNA integrated at the site of a chromosome break.

Previous analysis of plasmid DNA transfected into 108 cell clones demonstrated extensive polymorphism near the integration site in one clone. This polymorphism was apparent by Southern blot analysis as diffuse bands that extended over 30 kb. In the present study, nucleotide sequence analysis of cloned DNA from the integration site revealed telomere repeat sequences at the ends of the integrated plasmid DNA. The telomere repeat sequences at one end were located at the junction between the plasmid and cell DNA. The telomere repeat sequences at the other end were located in the opposite orientation in the polymorphic region and were shown by digestion with BAL 31 to be at the end of the chromosome. Telomere repeat sequences were not found at this location in the plasmid or parent cell DNA. Although the repeat sequences may have been acquired by recombination, a more likely explanation is that they were added to the ends of the plasmid by telomerase before integration. Comparison of the cell DNA before and after integration revealed that a chromosome break had occurred at the integration site, which was shown by fluorescent in situ hybridization to be located near the telomere of chromosome 13. These results demonstrate that chromosome breakage and rearrangement can result in interstitial telomere repeat sequences within the human genome. These sequences could promote genomic instability, because short repeat sequences can be recombinational hotspots. The results also show that DNA rearrangements involving telomere repeat sequences can be associated with chromosome breaks. The introduction of telomere repeat sequences at spontaneous or ionizing radiation-induced DNA strand breaks may therefore also be a mechanism of chromosome fragmentation.     

Comparison of repair of DNA double-strand breaks in identical sequences in primary human fibroblast and immortal hamster-human hybrid cells harboring a single copy of human chromosome 11

We have optimized a pulsed-field gel electrophoresis assay that measures induction and repair of double-strand breaks (DSBs) in specific regions of the genome (L?brich et al., Proc. Natl. Acad. Sci. USA 92, 12050-12054, 1995). The increased sensitivity resulting from these improvements makes it possible to analyze the size distribution of broken DNA molecules immediately after the introduction of DSBs and after repair incubation. This analysis shows that the distribution of broken DNA pieces after exposure to sparsely ionizing radiation is consistent with the distribution expected from randomly induced DSBs. It is apparent from the distribution of rejoined DNA pieces after repair incubation that DNA ends continue to rejoin between 3 and 24 h postirradiation and that some of these rejoining events are in fact misrejoining events, since novel restriction fragments both larger and smaller than the original fragment are generated after repair. This improved assay was also used to study the kinetics of DSB rejoining and the extent of misrejoining in identical DNA sequences in human GM38 cells and human-hamster hybrid A(L) cells containing a single human chromosome 11. Despite the numerous differences between these cells, which include species and tissue of origin, levels of TP53, expression of telomerase, and the presence or absence of a homologous chromosome for the restriction fragments examined, the kinetics of rejoining of radiation-induced DSBs and the extent of misrejoining were similar in the two cell lines when studied in the G(1) phase of the cell cycle. Furthermore, DSBs were removed from the single-copy human chromosome in the hamster A(L) cells with similar kinetics and misrejoining frequency as at a locus on this hybrid's CHO chromosomes.     

Sequence-specific DNA recognition by the myb-like domain of the human telomere binding protein TRF1: a model for the protein-DNA complex.

Telomeres consist of tandem arrays of short G-rich sequence motifs packaged by specific DNA binding proteins. In humans the double-stranded telomeric TTAGGG repeats are specifically bound by TRF1 and TRF2. Although telomere binding proteins from evolutionarily distant species are not sequence homologues, they share a Myb-like DNA binding motif. Here we have used gel retardation, primer extension and DNase I footprinting analyses to define the binding site of the isolated Myb-like domain of TRF1 and present a three-dimensional model for its interaction with human telomeric DNA. Our results suggest that the Myb-like domain of TRF1 recognizes a binding site centred on the sequence GGGTTA and that its DNA binding mode is similar to that of the homeodomain-like motifs of the yeast telomere binding protein RAP1. The implications of these findings for recognition of telomeric DNA in general are discussed.     

Endocytosis of insulin-like growth factor II by a mini-receptor based on repeat 11 of the mannose 6-phosphate/insulin-like growth factor II receptor

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) plays an important role in controlling the extracellular level of the insulin-like growth factor II (IGF-II) by mediating its binding at the cell surface and delivery to lysosomes. Loss of the receptor is associated with an accumulation of IGF-II, which can cause perinatal lethality if it is systemic, or local proliferation and tumorgenesis if it is spatially restricted. The extracytoplasmic domain of the receptor consists of 15 homologous repeats, of which repeat 11 carries the IGF-II-binding site of the multifunctional receptor. To investigate whether repeat 11 is sufficient to mediate binding and internalization of IGF-II, a construct consisting of repeat 11 fused to the transmembrane and cytoplasmic domain of the M6P/IGF-II receptor was transfected into mouse embryonic fibroblasts. The construct was expressed as a stable membrane protein which binds IGF-II with a 10-fold lower affinity as observed for the M6P/IGF-II receptor and is found at the cell surface and in endosomes. It mediates the internalization of IGF-II and its delivery to lysosomes, suggesting that it can function as a IGF-II mini-receptor controlling the extracellular IGF-II level.     

Chemiluminescence detection of telomere DNA in human cells on a membrane by using fluorescein-5-isothiocyanate-labeled primers

Telomere DNA is related to cell aging and cancer genesis because the telomeric region of DNA sequences at chromosome ends are shortened with cell divisions. Therefore, a sensitive and specific detection method is required for the telomere DNA. Here we propose a chemiluminescence (CL)-based method for the sensitive detection of telomere DNA in human cells. In this study, the telomere DNA was amplified by polymerase chain reaction (PCR) using special forward and reverse primers labeled with fluorescein-5-isothiocyanate (FITC) at the 5′ end, and then the FITC-containing PCR products were detected by CL reaction with 3,4,5-trimethoxyphenylglyoxal (TMPG) after electrophoresis followed by Southern blot onto a nylon membrane. The TMPG reagent specifically reacted with guanine moiety in DNA at room temperature and provided CL intensities. The CL intensities from the PCR products could be enhanced approximately 10-fold using FITC-labeled primers as compared with those using nonlabeled primers. The detection limit of the PCR products with the proposed method was 0.3 ng on the membrane. The developed CL method could quantitatively determine the telomere DNA in a small number of human cells (∼350) and gave approximately 10 times higher sensitivity than a conventional fluorescence-based method.     

Ultrafast electron transfer in the recognition of different DNA sequences by a DNA-binding protein with different dynamical conformations

Ultrafast electron transfer (ET) phenomenon in protein and protein-DNA complex is very much crucial and often leads to the regulation of various kinds of redox reactions in biological system. Although, the conformation of the protein in protein-DNA complex is concluded to play the key role in the ET process, till date very little evidences exist in the literature. λ-repressor-operator DNA interaction, particularly O(R)1 and O(R)2, is a key component of the λ-genetic switch and is a model system for understanding the chemical principles of the conformation-dependent ET reaction, governed by differential protein dynamics upon binding with different DNA target sequences. Here, we have explored the photoinduced electron transfer from the tryptophan moieties of the protein λ-repressor to two operators DNA of different sequences (O(R)1 and O(R)2) using picosecond-resolved fluorescence spectroscopy. The enhanced flexibility and different conformation of the C-terminal domain of the repressor upon complexation with O(R)1 DNA compared to O(R)2 DNA are found to have pronounced effect on the rate of ET. We have also observed the ET phenomenon from a dansyl chromophore, bound to the lysine residue, distal from the DNA-binding domain of the protein to the operator DNA with a specific excitation at 299 nm wavelength. The altered ET dynamics as a consequence of differential protein conformation upon specific DNA sequence recognition may have tremendous biological implications.     

Ultrafast electron transfer in the recognition of different DNA sequences by a DNA-binding protein with different dynamical conformations

Ultrafast electron transfer (ET) phenomenon in protein and protein–DNA complex is very much crucial and often leads to the regulation of various kinds of redox reactions in biological system. Although, the conformation of the protein in protein–DNA complex is concluded to play the key role in the ET process, till date very little evidences exist in the literature. λ-repressor–operator DNA interaction, particularly O_R1 and O_R2, is a key component of the λ-genetic switch and is a model system for understanding the chemical principles of the conformation-dependent ET reaction, governed by differential protein dynamics upon binding with different DNA target sequences. Here, we have explored the photoinduced electron transfer from the tryptophan moieties of the protein λ-repressor to two operators DNA of different sequences (O_R1 and O_R2) using picosecond-resolved fluorescence spectroscopy. The enhanced flexibility and different conformation of the C-terminal domain of the repressor upon complexation with O_R1 DNA compared to O_R2 DNA are found to have pronounced effect on the rate of ET. We have also observed the ET phenomenon from a dansyl chromophore, bound to the lysine residue, distal from the DNA-binding domain of the protein to the operator DNA with a specific excitation at 299?nm wavelength. The altered ET dynamics as a consequence of differential protein conformation upon specific DNA sequence recognition may have tremendous biological implications.     

Using multi-matching system based on a simplified deformable model of the human iris for iris recognition

1 Introduction Iris recognition technology is based on the stableand distinctive iris patterns, and has attracted a lot ofattention recently. Nowadays it has become a most im-portant biometric solution for personal identification. Since the 1990s, much work on iris recognition hasbeen done and great progress has been made. Some pre-dominant algorithms for iris recognition have beenproposed. Daugman generated an iris code by quantizingthe local phase angle using a set of 2-D Gabor f…     

Discovery and structure-guided drug design of inhibitors of 11β-hydroxysteroid-dehydrogenase type I based on a spiro-carboxamide scaffold

Spiro-carboxamides were identified as inhibitors of 11β-hydroxysteroid-dehydrogenase type 1 by high-throughput screening. Structure-based drug design was used to optimise the initial hit yielding a sub-nanomolar IC₅₀ inhibitor (0.5 nM) on human 11β-HSD1 with a high binding efficiency index (BEI of 32.7) which was selective against human 11β-HSD2 (selectivity ratio > 200000).     

Mapping of human plasma kallikrein active site by design of peptides based on modifications of a Kazal-type inhibitor reactive site

Human plasma kallikrein (huPK) is a proteinase that participates in several biological processes. Although various inhibitors control its activity, members of the Kazal family have not been identified as huPK inhibitors. In order to map the enzyme active site, we synthesized peptides based on the reactive site (PRILSPV) of a natural Kazal-type inhibitor found in Cayman plasma, which is not an huPK inhibitor. As expected, the leader peptide (Abz-SAPRILSPVQ-EDDnp) was not cleaved by huPK. Modifications to the leader peptide at P'1, P'3 and P'4 positions were made according to the sequence of a phage display-generated recombinant Kazal inhibitor (PYTLKWV) that presented huPK-binding ability. Novel peptides were identified as substrates for huPK and related enzymes. Both porcine pancreatic and human plasma kallikreins cleaved peptides at Arg or Lys bonds, whereas human pancreatic kallikrein cleaved bonds involving Arg or a pair of hydrophobic amino acid residues. Peptide hydrolysis by pancreatic kallikrein was not significantly altered by amino acid replacements. The peptide Abz-SAPRILSWVQ-EDDnp was the best substrate and a competitive inhibitor for huPK, indicating that Trp residue at the P'4 position is important for enzyme action.     

Development of a fungal transformation system based on selection of sequences with promoter activity.

A novel strategy was used to develop a transformation system for the plant pathogenic fungus Cochliobolus heterostrophus. Sequences capable of driving the expression of a gene conferring resistance to the antibiotic hygromycin B in C. heterostrophus were selected from a library of genomic DNA fragments and used, with the selectable marker, as the basis for transformation. The library of random 0.5- to 2.0-kilobase-pair fragments of C. heterostrophus genomic DNA was inserted at the 5' end of a truncated, promoterless Escherichia coli hygromycin B phosphotransferase gene (hygB) whose product confers resistance to hygromycin B. C. heterostrophus protoplasts were transformed with the library and selected for resistance. Resistant colonies arose at low frequency. Each colony contained a transformation vector stably integrated into chromosomal DNA. When the transforming DNA was recovered from the genome and introduced into C. heterostrophus, resistant colonies appeared at higher frequency. We determined the sequences of two of the C. heterostrophus DNA fragments which had been inserted at the 5' end of hygB in the promoter library and found that both made translational fusions with hygB. One of the two fusions apparently adds 65 and the other at least 86 amino acids to the N-terminus of the hygB product. Plasmids containing hygB-C. heterostrophus promoter fusions can be used unaltered to drive hygB expression in several other filamentous ascomycetes. This approach to achieving transformation may have general utility, especially for organisms with relatively undeveloped genetics.     

Development of mapped simple sequence repeat markers from common bean (Phaseolus vulgaris L.) based on genome sequences of a Chinese landrace and diversity evaluation

Microsatellite or single sequence repeat (SSR) markers have been commonly used in genetic research in many crop species, including common bean (Phaseolus vulgaris L.). A limited number of existing SSR markers have been designed from high-throughput sequencing of the genome, warranting the exploitation of new SSR markers from genomic regions. In this paper, we sequenced total DNA from the genotype Hong Yundou with a 454-FLX pyrosequencer and found numerous SSR loci. Based on these, a large number of SSR markers were developed and 90 genomic-SSR markers with clear bands were tested for mapping and diversity detection. The new SSR markers proved to be highly polymorphic for molecular polymorphism, with an average polymorphism information content value of 0.44 in 131 Chinese genotypes and breeding lines, effective for distinguishing Andean and Mesoamerican genotypes. In addition, we integrated 85 primers of the 90 polymorphism markers into the bean map using an F2 segregating population derived from Hong Yundou crossed with Jingdou. The distribution of SSR markers among 11 chromosomes was not random and tended to cluster on the linkage map, with 14 new markers mapped on chromosome Pv01, whereas only four loci were located on chromosome Pv04. Overall, these new markers have potential for genetic mapping, genetic diversity studies and map-based cloning in common bean.     

Non-reducing terminal fucose within N-linked glycan plays a significant role in the recognition of human milk lactoferrin by the 1CF11 monoclonal antibody

We have recently demonstrated that the 1CF11 monoclonal antibody bound human milk lactoferrin (hLf) through the recognition of two distinct portions of the molecule, namely the N-glycan-relevant and -irrelevant structural elements. In this present study, we prepared four immunoreactive peptide fractions containing N-linked glycan from tryptic digests of reduced and alkylated hLf by using a concanavalin A lectin column and reverse-phase HPLC. Deglycosylation of these fractions and a competitive binding assay using fucosylated oligosaccharides revealed that the non-reducing terminal fucose residue in N-linked glycan(s) played a significant role in recognizing the N-glycan-relevant element in hLf by 1CF11.     

PI16 is expressed by a subset of human memory Treg with enhanced migration to CCL17 and CCL20

It is well known that adoptive transfer of donor-derived tolerogenic dendritic cells (DCs) helps to induce immune tolerance. RelB, one of NF-κB subunits, is a critical element involved in DC maturation. In the present study, our results showed tolerogenic DCs could be acquired via silencing RelB using small interfering RNA. Compared with imDCs, the tolerogenic DCs had more potent ability to inhibit mixed lymphocyte reaction (MLR) and down-regulate Th1 cytokines and prompt the production of Th2 cytokines. They both mediated immune tolerance via the increased of T cell apoptosis and generation of regulatory T cells. Administration of donor-derived tolerogenic DCs significantly prevented the allograft rejection and prolonged the survival time in a murine heart transplantation model. Our results demonstrate donor-derived, RelB-shRNA induced tolerogenic DCs can significantly induce immune tolerance in vitro and in vivo.     

Isolation of DNA sequences on human chromosome 21 by application of a recombination-based assay to DNA from flow-sorted chromosomes

Summary By merging two efficient technologies, bivariate flow sorting of human metaphase chromosomes and a recombination-based assay for sequence complexity, we isolated 28 cloned DNA segments homologous to loci on human chromosome 21. Subregional mapping of these DNA segments with a somatic cell hybrid panel showed that 26 of the 28 cloned DNA sequences are distributed along the long arm of chromosome 21, while the other 2 hybridize with sequences on the short arm of both chromosome 21 and other chromosomes. This new collection of probes homologous to chromosome 21 should facilitate molecular analyses of trisomy 21 by providing DNA probes for the linkage map of chromosome 21, for studies of nondisjunction, for chromosome walking in clinically relevant subregions of chromosome 21, and for the isolation of genes on chromosome 21 following the screening of cDNA libraries.     

Direct prediction of profiles of sequences compatible with a protein structure by neural networks with fragment‐based local and energy‐based nonlocal profiles

Locating sequences compatible with a protein structural fold is the well‐known inverse protein‐folding problem. While significant progress has been made, the success rate of protein design remains low. As a result, a library of designed sequences or profile of sequences is currently employed for guiding experimental screening or directed evolution. Sequence profiles can be computationally predicted by iterative mutations of a random sequence to produce energy‐optimized sequences, or by combining sequences of structurally similar fragments in a template library. The latter approach is computationally more efficient but yields less accurate profiles than the former because of lacking tertiary structural information. Here we present a method called SPIN that predicts Sequence Profiles by Integrated Neural network based on fragment‐derived sequence profiles and structure‐derived energy profiles. SPIN improves over the fragment‐derived profile by 6.7% (from 23.6 to 30.3%) in sequence identity between predicted and wild‐type sequences. The method also reduces the number of residues in low complex regions by 15.7% and has a significantly better balance of hydrophilic and hydrophobic residues at protein surface. The accuracy of sequence profiles obtained is comparable to those generated from the protein design program RosettaDesign 3.5. This highly efficient method for predicting sequence profiles from structures will be useful as a single‐body scoring term for improving scoring functions used in protein design and fold recognition. It also complements protein design programs in guiding experimental design of the sequence library for screening and directed evolution of designed sequences. The SPIN server is available at http://sparks‐lab.org . Proteins 2014; 82:2565–2573. © 2014 Wiley Periodicals, Inc.