Structural and biophysical properties of h-FANCI ARM repeat protein

Fanconi anemia complementation groups – I (FANCI) protein facilitates DNA ICL (Inter-Cross-link) repair and plays a crucial role in genomic integrity. FANCI is a 1328 amino acids protein which contains armadillo (ARM) repeats and EDGE motif at the C-terminus. ARM repeats are functionally diverse and evolutionarily conserved domain that plays a pivotal role in protein–protein and protein–DNA interactions. Considering the importance of ARM repeats, we have explored comprehensive in silico and in vitro approach to examine folding pattern. Size exclusion chromatography, dynamic light scattering (DLS) and glutaraldehyde crosslinking studies suggest that FANCI ARM repeat exist as monomer as well as in oligomeric forms. Circular dichroism (CD) and fluorescence spectroscopy results demonstrate that protein has predominantly α- helices and well-folded tertiary structure. DNA binding was analysed using electrophoretic mobility shift assay by autoradiography. Temperature-dependent CD, Fluorescence spectroscopy and DLS studies concluded that protein unfolds and start forming oligomer from 30°C. The existence of stable portion within FANCI ARM repeat was examined using limited proteolysis and mass spectrometry. The normal mode analysis, molecular dynamics and principal component analysis demonstrated that helix-turn-helix (HTH) motif present in ARM repeat is highly dynamic and has anti-correlated motion. Furthermore, FANCI ARM repeat has HTH structural motif which binds to double-stranded DNA.     

ILPR repeats adopt diverse G‐quadruplex conformations that determine insulin binding

The insulin‐linked polymorphic region (ILPR) is a VNTR region located upstream of the insulin (INS) gene consisting of the repeat 5′‐ACAGGGGTGTGGGG (repeat a) and several less abundant sequence repeats (b–n). Here, we have investigated the structural polymorphism of G‐quadruplexes formed from the most common repeat sequences (a–c) and their effect on insulin protein binding. We first established that the ILPR repeats “b” and “c” can form quadruplex structures. Insulin has previously been shown to bind a G‐quadruplex formed by a dimer of the repeat “a”. Our findings show that insulin binds preferentially to the repeat “a” G‐quadruplex (K_d = 0.17 ± 0.03 μM) over G‐quadruplexes formed from other ILPR repeats that were tested (K_ds from 0.71 ± 0.15 to 1.07 ± 0.09 μM). Additionally, the Watson‐Crick complementary relationship between the loop regions of repeat “a” (ACA and TGT) seemingly play an important role in favoring a specific G‐quadruplex conformation, which based on our data is critical for insulin binding. Affinity for insulin is reduced in sequences lacking the putative WC complementarity, however upon engineered restoration of complementarity, insulin binding is recovered. A DMS footprinting assay on the repeat “a” G‐quadruplex in the presence of insulin, combined with binding affinities for ILPR mutants led to identification of a loop nucleotide critical for binding. Uniquely, insulin shows clear preference for binding to the G‐quadruplexes with the more antiparallel feature. Collectively, our results illustrate the specific nature of insulin binding to the ILPR G‐quadruplexes and begin to provide molecular details on such interactions. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 21–31, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Cloning and expression of ARMC3-v2, a novel splicing variant of human ARMC3 gene

ARM genes, whose polypeptide consist of Armadillo/beta-catenin-like repeats (ARM) domain(s), exist ubiquitously from flies to vertebrates. These genes have multiple functions in signal transduction, development, cell adhesion and mobility, tumor initiation and metastasis. In this study, we have isolated a 2439-bp novel splicing variant of ARMC3 from the human fetal brain, encoding a 688-amino acid polypeptide that contains three typical ARM domains. The cDNA named ARMC3_v2 and the original named ARMC3_v1 (Gen-Bank: BC039312) are both located on the human chromosome 10p12.23. RT-PCR analysis in our work showed that ARMC3_v2 was detected in human skeletal muscle, liver, spleen and thymus; in contrast, ARMC3_v1 in skeletal muscle, lung, prostate and testis. The text was submitted by the authors in English.     

The adoption of a twisted structure of importin-beta is essential for the protein-protein interaction required for nuclear transport

Importin-beta is a nuclear transport factor which mediates the nuclear import of various nuclear proteins. The N-terminal 1-449 residue fragment of mouse importin-beta (impbeta449) possesses the ability to bidirectionally translocate through the nuclear pore complex (NPC), and to bind RanGTP. The structure of the uncomplexed form of impbeta449 has been solved at a 2.6 A resolution by X-ray crystallography. It consists of ten copies of the tandemly arrayed HEAT repeat and exhibits conformational flexibility which is involved in protein-protein interaction for nuclear transport. The overall conformation of the HEAT repeats shows that a twisted motion produces a significantly varied superhelical architecture from the previously reported structure of RanGTP-bound importin-beta. These conformational changes appear to be the sum of small conformational changes throughout the polypeptide. Such a flexibility, which resides in the stacked HEAT repeats, is essential for interaction with RanGTP or with NPCs. Furthermore, it was found that impbeta449 has a structural similarity with another nuclear migrating protein, namely beta-catenin, which is composed of another type of helix-repeated structure of ARM repeat. Interestingly, the essential regions for NPC translocation for both importin-beta and beta-catenin are spatially well overlapped with one another. This strongly indicates the importance of helix stacking of the HEAT or ARM repeats for NPC-passage.     

Cloning and expression of ARMC3_v2, a novel splicing variant of the human ARMC3 gene

ARM genes, whose polypeptide consist of Armadillo/beta-catenin-like repeats (ARM) domain(s), exist ubiquitously from fly to vertebrates. These genes have multiple functions in signal transduction, development, cell adhesion and mobility, tumor initiation and metastasis. In this study, we have isolated a novel splicing variant of ARMC3 from human fetal brain, which is 2439 bp, encoding a 688-amino acid polypeptide that contains three typical ARM domains. The cDNA called ARMC3_v2 and the original called ARMC3_v1 (GeneBank: BC039312) are both located on the human chromosome 10p12.23. RT-PCR analysis in our work showed that ARMC3_v2 was detected in human skeletal muscle, liver, spleen and thymus; in contrast, ARMC3_v1 in skeletal muscle, lung, prostate and testis.     

A large complement of the predicted Arabidopsis ARM repeat proteins are members of the U-box E3 ubiquitin ligase family

The Arabidopsis genome was searched to identify predicted proteins containing armadillo (ARM) repeats, a motif known to mediate protein-protein interactions in a number of different animal proteins. Using domain database predictions and models generated in this study, 108 Arabidopsis proteins were identified that contained a minimum of two ARM repeats with the majority of proteins containing four to eight ARM repeats. Clustering analysis showed that the 108 predicted Arabidopsis ARM repeat proteins could be divided into multiple groups with wide differences in their domain compositions and organizations. Interestingly, 41 of the 108 Arabidopsis ARM repeat proteins contained a U-box, a motif present in a family of E3 ligases, and these proteins represented the largest class of Arabidopsis ARM repeat proteins. In 14 of these U-box/ARM repeat proteins, there was also a novel conserved domain identified in the N-terminal region. Based on the phylogenetic tree, representative U-box/ARM repeat proteins were selected for further study. RNA-blot analyses revealed that these U-box/ARM proteins are expressed in a variety of tissues in Arabidopsis. In addition, the selected U-box/ARM proteins were found to be functional E3 ubiquitin ligases. Thus, these U-box/ARM proteins represent a new family of E3 ligases in Arabidopsis.     

Do sequence repeats play an equivalent role in the choline-binding module of pneumococcal LytA amidase?

LytA amidase breaks down the N-acetylmuramoyl-l-alanine bonds in the peptidoglycan backbone of Streptococcus pneumoniae. Its polypeptide chain has two modules: the NH(2)-terminal module, responsible for the catalytic activity, and the COOH-terminal module, constructed by six tandem repeats of 20 or 21 amino acids (p1-p6) and a short COOH-terminal tail. The polypeptide chain must contain at least four repeats to efficiently anchor the autolysin to the choline residues of the cell wall. Nevertheless, the catalytic efficiency decreases by 90% upon deletion of the final tail. The structural implications of deleting step by step the two last (p5 and p6) repeats and the final COOH-tail and their effects on choline-amidase interactions have been examined by comparing four truncated mutants with LytA amidase by means of different techniques. Removal of this region has minor effects on secondary structure content but significantly affects the stability of native conformations. The last 11 amino acids and the p5 repeat stabilize the COOH-terminal module; each increases the module transition temperature by about 6 degrees C. Moreover, the p5 motif also seems to participate, in a choline-dependent way, in the stabilization of the NH(2)-terminal module. The effects of choline binding on the thermal stability profile of the mutant lacking the p5 repeat might reflect a cooperative pathway providing molecular communication between the choline-binding module and the NH(2)-terminal region. The three sequence motives favor the choline-amidase interaction, but the tail is an essential factor in the monomer <--> dimer self-association equilibrium of LytA and its regulation by choline. The final tail is required for preferential interaction of choline with LytA dimers and for the existence of different sets of choline-binding sites. The p6 repeat scarcely affects the amidase stability but could provide the proper three-dimensional orientation of the final tail.     

Extensive Amplification and Transposition of a Novel Repetitive Element, Xstir, Together with Its Terminal Inverted Repeat in the Evolution of Xenopus

A DNA fragment containing short tandem repeat sequences (approximately 86-bp repeat) was isolated from a Xenopus laevis cDNA library. Southern blot and in situ hybridization analyses revealed that the repeat was highly dispersed in the genome and was present at approximately 1 million copies per haploid genome. We named this element Xstir (Xenopus short tandemly and invertedly repeating element) after its arrangement in the genome. The majority of the genomic Xstir sequences were digested to monomer and dimer sizes with several restriction enzymes. Their sequences were found to be highly homogeneous and organized into tandem arrays in the genome. Alignment analyses of several known sequences showed that some of the Xstir-like sequences were also organized into interspersed inverted repeats. The inverted repeats consisted of an inverted pair of two differently modified Xstirs separated by a short insert. In addition, these were framed by another novel inverted repeat (Xstir-TIR). The Xstir-TIR sequence was also found at the ends of tandem Xstir arrays. Furthermore, we found that Xstir-TIR was linked to a motif characterizing the T2 family which belonged to a vertebrate MITE (miniature inverted-repeat transposable element) family, suggesting the importance of Xstir-TIR for their amplification and transposition. The present study of 11 anuran and 2 urodele species revealed that Xstir or Xstir-like sequences were extensively amplified in the three Xenopus species. Genomic Xstir populations of X. borealis and X. laevis were mutually indistinguishable but significantly different from that of X. tropicalis. Received: 5 April 2000 / Accepted: 3 August 2000     

Polypyrimidine tract‐binding protein is required for the repression of gene expression by all‐trans retinoic acid

All‐trans retinoic acid is a key regulator of early development. High concentrations of retinoic acid interfere with differentiation and migration of neural crest cells. Here we report that a dinucleotide repeat in the cis‐element of Snail2 (previously known as Slug) gene plays a role in repression by all‐trans retinoic acid. We analyzed the cis‐acting regulatory regions of the Xenopus Snail2 gene, whose expression is repressed by all‐trans retinoic acid. The analysis identified a TG/CA repeat as a necessary element for the repression. By performing a yeast one‐hybrid screen, we found that a polypyrimidine tract‐binding protein (PTB), which is known to be a regulator of the alternative splicing of pre‐messenger RNA, binds to the TG/CA repeat. Overexpression and knockdown experiments for PTB in HEK293 cells and Xenopus embryos indicated that PTB is required for repression by retinoic acid. The green fluorescent protein‐PTB fusion protein was localized in the nucleus of 293T cells. In situ hybridization for PTB in Xenopus embryos showed that PTB is expressed at the regions including neural crest at the early stages. Our results indicate that PTB plays a role in the repression of gene expression by retinoic acid through binding to the TG/CA repeats.     

Lineage-Specific Tandem Repeats Riding on a Transposable Element of MITE in <Emphasis Type="Italic">Xenopus</Emphasis> Evolution: A New Mechanism for Creating Simple Sequence Repeats

Xstir is a repetitive DNA sequence element that is extremely amplified as a common component of two different structures: a tandem repeat (Xstir array) and a MITE (miniature inverted-repeat transposable element) in the genome of Xenopus laevis. To elucidate the origin and evolutionary history of Xstir-related sequences, we investigated their species specificity among three Xenopus species (X. laevis, X. borealis, and X. tropicalis). Analyses by sequence alignment and digestion with restriction enzymes of genomic Xstir-related sequences revealed that the MITE (Xmix MITE) was well conserved among the three Xenopus species, with small lineage-specific differences. On the other hand, the tandem repeat element (tropXstir) in X. tropicalis was different from the Xstir that X. laevis and X. borealis have in common. Both sequences of Xstir and tropXstir were, however, different segments of the Xmix MITE. The results suggest that these tandem repeats were formed by partial tandem duplication of the MITE internal sequence in each lineage of X. tropicalis and of X. borealis/X. laevis after their branching. A molecular mechanism for creating and elongating the tandem repeats from the MITE is proposed.Reviewing Editor: Dr. Jerzy Jurka     

Geminivirus transmission and biological characterisation of Bemisia tabaci (Gennadius) biotypes from different geographic regions

Eighteen populations of Bemisia tabaci, collected from different geographic locations (North & Central America, the Caribbean, Africa, the Middle East, Asia and Europe), were studied to identify and compare biological and genetic characteristics that can be used to differentiate biotypes. The morphology of the fourth instar/pupal stage and compound eye structures of adults were investigated using scanning electron microscopy and found to be typical of the species among all biotypes and populations studied. Setae and spines of B. tabaci larval scales from the same colony were highly variable depending on the host plant species or leaf surface characteristics. The location and the morphology of caudal setae, characteristic of all B. tabaci studied to date, were present in all colonies. However, differences in adult body lengths and in the ability to induce phy to toxic disorders in certain plant species were found between biotypes or populations. The recently identified “B” biotype, characterised by a diagnostic esterase banding pattern and by its ability to induce phytotoxic responses in squash, honeysuckle and nightshade was readily distinguished from non-“B” biotype populations. None of the non-“B” biotypes studied, were found to induce phytotoxic responses. Nine populations examined showed typical “B” biotype characteristics, regardless of country of origin. All tested populations, determined as “B” or “B”-like biotypes successfully mated with other “B” biotype colonies from different geographic areas. Non-“B” biotype colonies did not interbreed with other biotypes. The B. tabaci populations were tested for their ability to transmit 15 whitefly-transmitted geminiviruses (WTGs) from different geographic areas with a wide range of symptom types. All WTGs were transmitted by the “B” biotype colonies and by most non-“B” biotype colonies, with the exception of three viruses found in ornamental plants which were non-transmissible by any colony. Some non-“B” biotypes would not transmit certain geminiviruses and some geminiviruses were more efficiently transmitted than were others.     

SadA,a novel adhesion receptor in Dictyostelium

Little is known about cell-substrate adhesion and how motile and adhesive forces work together in moving cells. The ability to rapidly screen a large number of insertional mutants prompted us to perform a genetic screen in Dictyostelium to isolate adhesion-deficient mutants. The resulting substrate adhesion-deficient (sad) mutants grew in plastic dishes without attaching to the substrate. The cells were often larger than their wild-type parents and displayed a rough surface with many apparent blebs. One of these mutants, sadA-, completely lacked substrate adhesion in growth medium. The sadA- mutant also showed slightly impaired cytokinesis, an aberrant F-actin organization, and a phagocytosis defect. Deletion of the sadA gene by homologous recombination recreated the original mutant phenotype. Expression of sadA-GFP in sadA-null cells restored the wild-type phenotype. In sadA-GFP-rescued mutant cells, sadA-GFP localized to the cell surface, appropriate for an adhesion molecule. SadA contains nine putative transmembrane domains and three conserved EGF-like repeats in a predicted extracellular domain. The EGF repeats are similar to corresponding regions in proteins known to be involved in adhesion, such as tenascins and integrins. Our data combined suggest that sadA is the first substrate adhesion receptor to be identified in Dictyostelium.     

High-throughput sequencing uncover Ficus tikoua Bur. chloroplast genome

Ficus tikoua Bur., called “di guo”, is a very important economic plant. However, we know little on its molecular mechanisms for a long time. In this study, three F. tikoua chloroplast genomes were first obtained through Illumina sequencing. The F. tikoua Bur. cp genomes exhibited a typical circular structure, including a pair of inverted repeats, a small single copy sequence, and a long single copy sequence. More than 100 genes and about 70 ncRNAs (rRNAs, tRNAs, snRNA, snoRNA, etc.) were identified in each cp genomes. Some repeats, including DNA element, long terminal repeat, small RNA, simple repeat and low complexity, were examined in the three complete cp genomes. In addition, phylogeny tree from six Ficus. species and five Morus. species in family Moracea were constructed for clarifying taxonomy. This data can help further the understanding of evolution and phylogenetic relationships in F. tikoua Bur.

     

Conformational energy analysis of the leucine repeat regions of C/EBP,GCN4, and the proteins of themyc,jun, andfos oncogenes

It has been recently proposed that certain DNA binding proteins (including C/EBP, GCN4 and themyc, jun, andfos oncogene proteins) share a common structural motif based on helix-promoting regions containing heptad repeat sequences of leucines. It has been suggested that this structure is critical to the biological activity of these proteins, since it facilitates the formation of functional dimers held together by interdigitating leucine side-chains along the hydrophobic interfaces between long α-helical regions of the polypeptide chains in a configuration termed the “leucine zipper.” In this paper, conformational energy analysis is used to determine the preferred three-dimensional structures of the leucine repeat regions of these proteins. The results indicate that, in all cases, the global minimum energy conformation for these regions is an amphipathic α-helix with the leucine side-chains arrayed on one side in such a way to favor “leucine zipper” dimerization. Furthermore, amino acid substitutions in these regions (such as Pro for Leu), that are known to inhibit dimer formation and prevent DNA binding, are found to produce significant conformational changes that disrupt the amphipathic helical structure. Thus, these results provide support for the proposed “leucine zipper” configuration as a critical structural feature of this class of DNA binding proteins.

     

JP-3 gene polymorphism in a healthy population of Serbia and Montenegro

Expansions of CTG repeats inJP-3 gene are associated with a phenotype similar to Huntington disease. These expansions are the cause of Huntington disease like-2 (HDL-2) phenotype. CTG repeats inJP-3 gene are polymorphic in healthy population. Analyses of CTG repeat polymorphism ofJP-3 gene in various healthy populations could help in estimating the population at risk for developing HDL-2. CTG repeat polymorphism ofJP-3 gene was analysed in healthy population of Serbia and Montenegro. Study included 198 unrelated subjects. Analyses ofJP-3 locus were performed using PCR and sequencing. Six differentJP-3 alleles were obtained and they were in the range of 11 to 18 CTG repeats showing a bimodal distribution, with peaks at 14 and 16. Results show that the distribution ofJP-3 alleles in population of Serbia and Montenegro is consistent with distributions in other analysed populations. The absence of alleles with more then 18 CTG repeats suggests that HDL-2 is very rare in the populations of Serbia and Montenegro.     

Planar and vertical induction of anteroposterior pattern during the development of the amphibian central nervous system

In amphibians and other vertebrates, neural development is induced in the ectoderm by signals coming from the dorsal mesoderm during gastrulation. Classical embryological results indicated that these signals follow a “vertical” path, from the involuted dorsal mesoderm to the overlying ectoderm. Recent work with the frog Xenopus laevis, however, has revealed the existence of “planar” neural-inducing signals, which pass within the continuous sheet or plane of tissue formed by the dorsal mesoderm and presumptive neurectoderm. Much of this work has made use of Keller explants, in which dorsal mesoderm and ectoderm are cultured in a planar configuration with contact along only a single edge, and vertical contact is prevented. Planar signals can induce the full anteroposterior (A-P) extent of neural pattern, as evidenced in Keller explants by the expression of genes that mark specific positions along the A-P axis. In this review, classical and modern molecular work on vertical and planar inductionwill be discussed. This will be followed by a discussion of various models for vertical induction and planar induction. It has been proposed that the A-P pattern in the nervous system is derived from a parallel pattern of inducers in the dorsal mesoderm which is “imprinted” vertically onto the overlying ectoderm. Since it is now known that planar signals can also induce A-P neural pattern, this kind of model must be reassessed. The study of planar induction of A-P pattern in Xenopus embryos provides a simple, manipulable, two-dimensional system in which to investigate pattern formation. © 1993 John Wiley & Sons, Inc.     

Identification of genes containing expanded purine repeats in the human genome and their apparent protective role against cancer

Purine repeat sequences present in a gene are unique as they have high propensity to form unusual DNA-triple helix structures. Friedreich’s ataxia is the only human disease that is well known to be associated with DNA-triplexes formed by purine repeats. The purpose of this study was to recognize the expanded purine repeats (EPRs) in human genome and find their correlation with cancer pathogenesis. We developed “PuRepeatFinder.pl” algorithm to identify non-overlapping EPRs without pyrimidine interruptions in the human genome and customized for searching repeat lengths, n ≥ 200. A total of 1158 EPRs were identified in the genome which followed Wakeby distribution. Two hundred and ninety-six EPRs were found in geneic regions of 282 genes (EPR-genes). Gene clustering of EPR-genes was done based on their cellular function and a large number of EPR-genes were found to be enzymes/enzyme modulators. Meta-analysis of 282 EPR-genes identified only 63 EPR-genes in association with cancer, mostly in breast, lung, and blood cancers. Protein–protein interaction network analysis of all 282 EPR-genes identified proteins including those in cadherins and VEGF. The two observations, that EPRs can induce mutations under malignant conditions and that identification of some EPR-gene products in vital cell signaling-mediated pathways, together suggest the crucial role of EPRs in carcinogenesis. The new link between EPR-genes and their functionally interacting proteins throws a new dimension in the present understanding of cancer pathogenesis and can help in planning therapeutic strategies. Validation of present results using techniques like NGS is required to establish the role of the EPR genes in cancer pathology.     

Two integrated partial repeats of simian virus 40 together code for a super-T antigen.

We determined that the coding sequence for a 100-kilodalton super-T antigen found in Simian virus 40 mouse transformants spanned two separate partial repeats of the viral genome. The downstream repeat contained a complete Simian virus 40 large-T-antigen gene, whereas the upstream repeat was a truncated copy of the same gene. When the repeats were separated by subcloning, the capacity to code for the super-T antigen was lost. A small insertion or deletion in the origin-control region which preceded the second repeat could also destroy the ability to code for the 100-kilodalton protein. Our data suggest that differential splicing between parts of two gene copies was responsible for the additional molecular weight of this super-T antigen.     

Tandemly Repeated Sequence Families in Micronuclear DNA of the Ciliate Stylonychia pustulata1

One third of a collection of cloned Stylonychia pustulata micronuclear DNA PstI fragments were found to be of a similar size, consistent with their being members of a repetitious sequence family with a repeat size of about 160 base pairs. Cross-hybridization experiments confirmed that these small cloned fragments are related by sequence homology. Hybridization of the cloned repetitious sequences to PstI digested micronuclear DNA revealed a “ladder” of bands (step size = 160 base pairs), indicating that the repeats are found in tandem arrays. This is the first demonstration of highly repetitious, tandemly repeated sequences in a ciliated protozoan.     

p62cdc23 of Saccharomyces cerevisiae: a nuclear tetratricopeptide repeat protein with two mutable domains.

CDC23 is required in Saccharomyces cerevisiae for cell cycle progression through the G2/M transition. The CDC23 gene product contains tandem, imperfect repeats, termed tetratricopeptide repeats, (TPR) units common to a protein family that includes several other nuclear division CDC genes. In this report we have used mutagenesis to probe the functional significance of the TPR units within CDC23. Analysis of truncated derivatives indicates that the TPR block of CDC23 is necessary for the function or stability of the polypeptide. In-frame deletion of a single TPR unit within the repeat block proved sufficient to inactivate CDC23 in vivo, though this allele could rescue the temperature-sensitive defect of a cdc23 point mutant by intragenic complementation. By both in vitro and in vivo mutagenesis techniques, 17 thermolabile cdc23 alleles were produced and examined. Fourteen alleles contained single amino acid changes that were found to cluster within two distinct mutable domains, one of which encompasses the most canonical TPR unit found in CDC23. In addition, we have characterized CDC23 as a 62-kDa protein (p62cdc23) that is localized to the yeast nucleus. Our mutagenesis results suggest that TPR blocks form an essential domain within members of the TPR family.