Extensive Deletions Research Articles

Spontaneous immunological targeting of endogenous retrovirus K is directed against the envelope protein in rhesus macaques

Endogenous retroviruses (ERVs), remnants of ancient retroviral infections, have invaded the germ line of every vertebrate species including both non-human primates and humans. Most ERVs are functionally crippled by extensive deletions, mutations and hypermethylation, leading to the view that they are inert genomic fossils. However, some ERVs retain the potential for biological activity and can produce mRNA transcripts, functional viral proteins, and even non-infectious virus particles during certain developmental and pathological processes. Although ERV gene products are implicated in the etiology of disease, their impact during host infection with exogenous retroviruses such as human and simian immunodeficiency virus (HIV/ SIV) remains unknown. Here, we identify the homologue of the biologically active human ERV-K, macaque ERV-K (macERV-K), which retainsintact open reading frames in the macaque genome. Indeed, we isolated a fully-spliced mRNA product encoding macERV-K Env, which contains all the structural features of a canonical retroviral Envelope protein. Furthermore, we identified robust CD8+ and CD4+ T cell responses directed at macERV-K Env in one SIV-infected Indian rhesus macaque. The macERV-K Envspecific CD4+ T cell response in this animal was larger than the entire 5lV-specific CD4+ T cell response and had a uniform effector memory phenotype, indicating continual low-level antigen exposure. In support of this, we also identified antibody responses targeting macERV-K in this animal. Although SIV infection did not appear to trigger ERV Env-specific T cell responses, these data suggest that Env proteins from ERVs can be active during infections with exogenous retroviruses. The interaction of ERV Env proteins expressed in HIV-infected cells may have major implications for the pathogenicity of HIV and warrants further investigation.

Abstract 5599: Screening for large genomic rearrangements in the FANCA and FANCJ genes reveals extensive genomic FANCA deletion in a Finnish breast cancer family

Abstract A portion of familial breast cancer is accounted for by mutations in the same genes that are inactivated in Fanconi anemia (FA). In the current study we set out to evaluate the role of large genomic rearrangements in the FANCA and FANCJ/BRIP1 genes in Finnish breast cancer families. Mutations in the upstream FANCA gene account for the majority of FA patients, and about one-third of all FANCA mutations are large intragenic deletions. Based on the gene's hypermutable nature, the protein's interaction with BRCA1 and its involvement in genomic integrity maintenance, we considered FANCA as a candidate for breast cancer susceptibility. In FANCJ, which is a downstream FA and known breast cancer susceptibility gene, early truncating mutations have been reported, but no large deletions have been recognized so far. For the present study, blood DNA from affected index persons of 100 Northern Finnish breast cancer families were assessed for possible constitutional exonic deletions or amplifications in FANCA and FANCJ by using the multiplex ligation-dependent probe amplification (MLPA) method. We identified a novel large heterozygous FANCA deletion in one of the studied breast cancer families, removing the promoter region and the first 12 exons of the gene. The defective allele was absent from all of the tested 124 controls as well as 540 unselected breast cancer cases, but was present in the father of the index patient having pancreatic cancer. No large deletions in FANCJ were uncovered. We conclude that large FANCA deletions might contribute to breast cancer susceptibility, therefore more extensive studies on their involvement in the disease are needed. To our knowledge, this is the first report on the existence of a large-size genomic deletion in an upstream FA gene in association with familial breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5599. doi:10.1158/1538-7445.AM2011-5599

The Trypanosoma cruzi Sylvio X10 strain maxicircle sequence: the third musketeer

BackgroundChagas disease has a diverse pathology caused by the parasite Trypanosoma cruzi, and is indigenous to Central and South America. A pronounced feature of the trypanosomes is the kinetoplast, which is comprised of catenated maxicircles and minicircles that provide the transcripts involved in uridine insertion/deletion RNA editing. T. cruzi exchange genetic material through a hybridization event. Extant strains are grouped into six discrete typing units by nuclear markers, and three clades, A, B, and C, based on maxicircle gene analysis. Clades A and B are the more closely related. Representative clade B and C maxicircles are known in their entirety, and portions of A, B, and C clades from multiple strains show intra-strain heterogeneity with the potential for maxicircle taxonomic markers that may correlate with clinical presentation.ResultsTo perform a genome-wide analysis of the three maxicircle clades, the coding region of clade A representative strain Sylvio X10 (a.k.a. Silvio X10) was sequenced by PCR amplification of specific fragments followed by assembly and comparison with the known CL Brener and Esmeraldo maxicircle sequences. The clade A rRNA and protein coding region maintained synteny with clades B and C. Amino acid analysis of non-edited and 5'-edited genes for Sylvio X10 showed the anticipated gene sequences, with notable frameshifts in the non-edited regions of Cyb and ND4. Comparisons of genes that undergo extensive uridine insertion and deletion display a high number of insertion/deletion mutations that are likely permissible due to the post-transcriptional activity of RNA editing.ConclusionPhylogenetic analysis of the entire maxicircle coding region supports the closer evolutionary relationship of clade B to A, consistent with uniparental mitochondrial inheritance from a discrete typing unit TcI parental strain and studies on smaller fragments of the mitochondrial genome. Gene variance that can be corrected by RNA editing hints at an unusual depth for maxicircle taxonomic markers, which will aid in the ability to distinguish strains, their corresponding symptoms, and further our understanding of the T. cruzi population structure. The prevalence of apparently compromised coding regions outside of normally edited regions hints at undescribed but active mechanisms of genetic exchange.

Screening for large genomic rearrangements in the FANCA gene reveals extensive deletion in a Finnish breast cancer family

A portion of familial breast cancer cases are caused by mutations in the same genes that are inactivated in the downstream part of Fanconi anemia (FA) signaling pathway. Here we have assessed the FANCA gene for breast cancer susceptibility by examining blood DNA for aberrations from 100 Northern Finnish breast cancer families using the MLPA method. We identified a novel heterozygous deletion, removing the promoter and 12 exons of the gene in one family. This allele was absent from 124 controls. We conclude that FANCA deletions might contribute to breast cancer susceptibility, potentially in combination with other germline mutations. To our knowledge, this is the first study reporting a large deletion in an upstream FA gene in familial breast cancer.

Web-based synoptic reporting for cancer checklists

Background: The surgical pathology report remains the primary source for information to guide the treatment of patients with cancer. Failure to report critical elements in a cancer report is an increasing problem in pathology because of the heightened complexity of these reports and number of elements that are important for patient care. The American College of Surgeons Commission on Cancer (ACS-CoC) in concert with the College of American Pathologists (CAP) developed checklists that contain all of the scientifically validated data elements that are to be reported for cancer specimens. Most institutions do not as of yet have pathology information systems in which CAP checklists are embedded into the laboratory information system (LIS). Entering the required elements often requires extensive text editing, secretarial support and deletion of extraneous elements that can be an arduous task.Materials and Methods: We sought to develop a web-based system that was available throughout the workstations in our department and was capable of generating synoptic reports based on the CAP guidelines. The program was written in a manner that allowed automatic generation of the web-based checklists through a parsing algorithm.Results: Multiple web-based synoptic report generators have been developed to encompass required elements of cancer synoptic reports as required by the ACS-CoC/CAP. In addition, utilizing the same program, report generators for certain molecular tests (KRAS mutation) and FISH studies (UroVysiontm) have also been developed. The output of these reports can be cut-and-pasted into any text-based anatomic pathology LIS. In addition, the elements can be compiled in a database.Conclusions: We describe a simple method to automate the development of web-based synoptic reports that can be entered into the anatomic pathology LIS and database.

New Determination Method for Extensive Gene Deletions In Diamond–Blackfan Anemia

AbstractAbstract 4231 Introduction:Fifty percent of Diamond–Blackfan anemia (DBA) patients possess mutations in ribosomal protein genes. Although several ribosomal protein genes, RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26, have been reported to be mutated in some DBA patients, including point mutations, nonsense mutations, deletions, splice site mutations, and translocations, other DBA patients appear to have intact ribosomal protein genes. To identify new mutations in ribosomal protein genes from a different aspect, we focused on extensive deletions in these genes, such as mutations involving loss of a whole allele. In this study, we applied quantitative genomic PCR, and successfully developed a convenient method for detecting extensive deletions designated the “DBA gene copy number assay”. Methods:DBA patients should have an intact allele and a mutated allele for the responsible ribosomal protein gene, meaning that they will have an abnormal karyotype (gene copy number of N) if they have an extensive deletion. We attempted to clarify the copy numbers of ribosomal protein genes by the difference in a 1-cycle delay of threshold in a quantitative PCR (q-PCR) assay. To detect extensive deletions, at least 2 sets of gene-specific primers for each DBA responsible gene (RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26) were prepared. Appropriate primers to fit the setting that the threshold cycle (Ct) of the q-PCR should occur within 1 cycle of the Ct scores of other primer sets were selected. After validation, we identified 6, 3, 4, 3, 3, 6, 9, 3, and 2 specific primer sets for RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26, respectively. By simply looking at the q-PCR amplification curves by eye, we were easily able to judge the copy numbers of 2N (normal) or N (abnormal) for the ribosomal protein genes. Results: We performed the DBA gene copy number assay for 14 randomly selected undiagnosed patients from the Japanese DBA genomic resource at the University of Hirosaki, who had no mutations by genomic sequencing analyses. For each case, all the DBA responsible genes were confirmed using the diagnostic primers. The results of the DBA gene copy number assays revealed that 5 of the 14 probands (36%) had an extensive deletion in one of the DBA responsible genes. As an interesting case among the 5 positive cases, we confirmed an extensive deletion in the RPS19 gene. The Ct scores for 4 of the 9 primer sets for RPS19 demonstrated a 1-cycle delay, while the scores for the other 5 primer sets were normal. By genomic PCR amplification analyses, we identified a deletion from nt. -1400 to +5757 (7157 nucleotides) in the RPS19 gene. The deleted region included the promoter region, and exons 1, 2, and 3 of the RPS19 gene. The remaining 4 cases were 1 proband with an RPL5 deletion, 1 with an RPL35A deletion and 2 with RPS17 deletions. In particular, the extensive deletions in the RPL5 and RPS17 alleles are the first such cases reported. Discussion:Since it has been difficult to address the loss of a whole allele in DBA, such mutations have not been precisely examined within the DBA responsible genes. Our data suggest that extensive deletions in ribosomal protein genes comprise a significant proportion of DBA cases in Japan. Our novel method could become a useful tool for screening the gene copy numbers of ribosomal protein genes, and for identifying new pathological mutations. Disclosures:No relevant conflicts of interest to declare.

Cpkk1, MAPKK of Cryphonectria parasitica, Is Necessary for Virulence on Chestnut

ABSTRACT The role of Cpkk1, a mitogen-activated protein kinase from Cryphonectria parasitica, was investigated by generating a number of mutant strains that overexpress, under the control of the cryparin promoter, both the wild-type protein and its allele with an extensive deletion in the catalytic domain. Furthermore, a hairpin construct was built and expressed to cause specific silencing of Cpkk1 mRNA transcripts. Specific mRNA silencing or overexpression was confirmed on both Northern and Western blot analysis. Selected C. parasitica strains with Cpkk1 either silenced or overexpressed were evaluated for their biological characteristics, including virulence on European chestnut, growth on different substrates, conidial sporulation, and resistance to cell-wall-degrading enzymes. Silencing of Cpkk1 and the overexpression of a defective Cpkk1 correlated with a marked reduction in virulence on 3-year-old chestnut trees, with no statistically significant effect on fungal growth in the various conditions tested.

Efficiency of nonhomologous DNA end joining varies among somatic tissues, despite similarity in mechanism.

Failure to repair DNA double-strand breaks (DSBs) can lead to cell death or cancer. Although nonhomologous end joining (NHEJ) has been studied extensively in mammals, little is known about it in primary tissues. Using oligomeric DNA mimicking endogenous DSBs, NHEJ in cell-free extracts of rat tissues were studied. Results show that efficiency of NHEJ is highest in lungs compared to other somatic tissues. DSBs with compatible and blunt ends joined without modifications, while noncompatible ends joined with minimal alterations in lungs and testes. Thymus exhibited elevated joining, followed by brain and spleen, which could be correlated with NHEJ gene expression. However, NHEJ efficiency was poor in terminally differentiated organs like heart, kidney and liver. Strikingly, NHEJ junctions from these tissues also showed extensive deletions and insertions. Hence, for the first time, we show that despite mode of joining being generally comparable, efficiency of NHEJ varies among primary tissues of mammals.

Identification of a Coiled Coil in Werner Syndrome Protein That Facilitates Multimerization and Promotes Exonuclease Processivity

Werner syndrome (WS) is a rare progeroid disorder characterized by genomic instability, increased cancer incidence, and early onset of a variety of aging pathologies. WS is unique among early aging syndromes in that affected individuals are developmentally normal, and phenotypic onset is in early adulthood. The protein defective in WS (WRN) is a member of the large RecQ family of helicases but is unique among this family in having an exonuclease. RecQ helicases form multimers, but the mechanism and consequence of multimerization remain incompletely defined. Here, we identify a novel heptad repeat coiled coil region between the WRN nuclease and helicase domains that facilitates multimerization of WRN. We mapped a novel and unique DNA-dependent protein kinase phosphorylation site proximal to the WRN multimerization region. However, phosphorylation at this site affected neither exonuclease activity nor multimeric state. We found that WRN nuclease is stimulated by DNA-dependent protein kinase independently of kinase activity or WRN nuclease multimeric status. In addition, WRN nuclease multimerization significantly increased nuclease processivity. We found that the novel WRN coiled coil domain is necessary for multimerization of the nuclease domain and sufficient to multimerize with full-length WRN in human cells. Importantly, correct homomultimerization is required for WRN function in vivo as overexpression of this multimerization domain caused increased sensitivity to camptothecin and 4-nitroquinoline 1-oxide similar to that in cells lacking functional WRN protein.

Hyperpolarization-activated inward leakage currents caused by deletion or mutation of carboxy-terminal tyrosines of the Na+/K+-ATPase α subunit

The Na+/K+-ATPase mediates electrogenic transport by exporting three Na+ ions in exchange for two K+ ions across the cell membrane per adenosine triphosphate molecule. The location of two Rb+ ions in the crystal structures of the Na+/K+-ATPase has defined two “common” cation binding sites, I and II, which accommodate Na+ or K+ ions during transport. The configuration of site III is still unknown, but the crystal structure has suggested a critical role of the carboxy-terminal KETYY motif for the formation of this “unique” Na+ binding site. Our two-electrode voltage clamp experiments on Xenopus oocytes show that deletion of two tyrosines at the carboxy terminus of the human Na+/K+-ATPase α2 subunit decreases the affinity for extracellular and intracellular Na+, in agreement with previous biochemical studies. Apparently, the ΔYY deletion changes Na+ affinity at site III but leaves the common sites unaffected, whereas the more extensive ΔKETYY deletion affects the unique site and the common sites as well. In the absence of extracellular K+, the ΔYY construct mediated ouabain-sensitive, hyperpolarization-activated inward currents, which were Na+ dependent and increased with acidification. Furthermore, the voltage dependence of rate constants from transient currents under Na+/Na+ exchange conditions was reversed, and the amounts of charge transported upon voltage pulses from a certain holding potential to hyperpolarizing potentials and back were unequal. These findings are incompatible with a reversible and exclusively extracellular Na+ release/binding mechanism. In analogy to the mechanism proposed for the H+ leak currents of the wild-type Na+/K+-ATPase, we suggest that the ΔYY deletion lowers the energy barrier for the intracellular Na+ occlusion reaction, thus destabilizing the Na+-occluded state and enabling inward leak currents. The leakage currents are prevented by aromatic amino acids at the carboxy terminus. Thus, the carboxy terminus of the Na+/K+-ATPase α subunit represents a structural and functional relay between Na+ binding site III and the intracellular cation occlusion gate.

Fusion of short telomeres in human cells is characterized by extensive deletion and microhomology, and can result in complex rearrangements

Telomere fusion is an important mutational event that has the potential to lead to large-scale genomic rearrangements of the types frequently observed in cancer. We have developed single-molecule approaches to detect, isolate and characterize the DNA sequence of telomere fusion events in human cells. Using these assays, we have detected complex fusion events that include fusion with interstitial loci adjacent to fragile sites, intra-molecular rearrangements, and fusion events involving the telomeres of both arms of the same chromosome consistent with ring chromosome formation. All fusion events were characterized by the deletion of at least one of the telomeres extending into the sub-telomeric DNA up to 5.6 kb; close to the limit of our assays. The deletion profile indicates that deletion may extend further into the chromosome. Short patches of DNA sequence homology with a G:C bias were observed at the fusion point in 60% of events. The distinct profile that accompanies telomere fusion may be a characteristic of the end-joining processes involved in the fusion event.

Genome Sequence of the Endosymbiont Rickettsia peacockii and Comparison with Virulent Rickettsia rickettsii: Identification of Virulence Factors

Rickettsia peacockii, also known as the East Side Agent, is a non-pathogenic obligate intracellular bacterium found as an endosymbiont in Dermacentor andersoni ticks in the western USA and Canada. Its presence in ticks is correlated with reduced prevalence of Rickettsia rickettsii, the agent of Rocky Mountain Spotted Fever. It has been proposed that a virulent SFG rickettsia underwent changes to become the East Side Agent. We determined the genome sequence of R. peacockii and provide a comparison to a closely related virulent R. rickettsii. The presence of 42 chromosomal copies of the ISRpe1 transposon in the genome of R. peacockii is associated with a lack of synteny with the genome of R. rickettsii and numerous deletions via recombination between transposon copies. The plasmid contains a number of genes from distantly related organisms, such as part of the glycosylation island of Pseudomonas aeruginosa. Genes deleted or mutated in R. peacockii which may relate to loss of virulence include those coding for an ankyrin repeat containing protein, DsbA, RickA, protease II, OmpA, ScaI, and a putative phosphoethanolamine transferase. The gene coding for the ankyrin repeat containing protein is especially implicated as it is mutated in R. rickettsii strain Iowa, which has attenuated virulence. Presence of numerous copies of the ISRpe1 transposon, likely acquired by lateral transfer from a Cardinium species, are associated with extensive genomic reorganization and deletions. The deletion and mutation of genes possibly involved in loss of virulence have been identified by this genomic comparison. It also illustrates that the introduction of a transposon into the genome can have varied effects; either correlating with an increase in pathogenicity as in Francisella tularensis or a loss of pathogenicity as in R. peacockii and the recombination enabled by multiple transposon copies can cause significant deletions in some genomes while not in others.

Abstract A216: Distinct peptides derived from the cochaperone Bag1-L inhibit prostate cancer cell growth

Abstract Introduction and objectives: Bag1 (Bcl-2 associated athanogene-1) is a family of proteins conserved throughout evolution, promoting cell survival and inhibiting apoptosis under stressful conditions. They are found overexpressed in several types of tumors and associated with poor prognosis. Intriguingly, the nuclear isoform of the Bag-1 proteins, Bag-1L, also exhibits pro-apoptotic properties through an unknown mechanism. In extensive deletion mutagenesis studies we identified sequences in the Bag-1L protein that exert pro-apoptotic action. The objective of the study is to characterize the mechanism of action of these sequences and to explore their use as candidate peptides for prostate cancer therapy. Materials and Methods: In vitro inhibition of tumor cell growth was analyzed in clonogenic assay where prostate cells were transfected with plasmids expressing different Bag-1L peptides. In vivo growth inhibition was analysed by the injection of prostate cells stably expressing the Bag-1L peptides into the flank of athymic nude mice and analyzing their effect on tumor size in vivo. Histological analysis for apoptosis was carried out on the tumor samples. For analyzing the interaction partners of the Bag-1L peptides, the peptides were generated as fusion proteins with glutathione S transferase (GST), immobilized on glutathione agarose beads and allowed to interact with lysates from prostate cells. Specifically bound proteins were then eluted and analysed by mass spectrometry and by Western blotting. Results: Overexpression of a 68 amino acid sequence of Bag-1L reduced the growth of 22Rv.1 prostate cancer cells but not of benign prostate hyperplasia cells in a clonogenic assay. Stable overexpression of this peptide in the 22Rv.1 prostate cancer cells also reduced cell growth in vivo in athymic nude mice. Histological analysis revealed massive apoptosis in the tumor cells expressing the peptide. Further deletion analysis of the 68 amino acid peptide showed that the N-terminal 40 amino acids but not the C-terminal 29 amino acids are sufficient for the growth inhibitory action. Protein-protein interaction studies using GST-pull down assay demonstrated that the growth inhibitory function of the peptides correlated with their ability to bind glucose regulated proteins 75 and 78 (GRP75 and GRP78). GRP75 and GRP78 have been implicated in tumor formation through their action in the mitochondria and in the endoplasmic reticulum in the regulation of the unfolded protein response. Conclusions: We have therefore identified Bag1-L peptides which when expressed in prostate tumor cells reduce the growth in vitro as well as in a mouse model of tumor formation. The action of the peptides is to induce apoptosis and it is correlated with their ability to bind the molecular chaperones GRP75 and GRP78. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A216.

Characterization of San Miguel Sea Lion Virus populations using pyrosequencing-based methods

San Miguel Sea Lion Virus (SMSV) is a small RNA virus in the genus Vesivirus with an unusually broad host range. Three populations of SMSV were examined by PCR amplification of the capsid precursor and putative helicase genes, followed by pyrosequencing. The populations were nasal swabs from two SMSV infected California sea lions (Zalophus californianus) from two different years, and a virus isolate from the earlier swab that was passaged in cell culture five times. In the capsid precursor, extensive deletions were prevalent in the passaged virus but uncommon in the clinical samples. A greater prevalence of point mutations was seen in the capsid precursor gene than in the putative helicase gene. In culture, the minority sequence in the capsid precursor at nucleotide position 5826 rapidly shifted after five passages to become the majority sequence. Levels of diversity at individual sites showed much more similarity between the two clinical samples than between the earlier clinical sample and the passaged culture from the same sample. SMSV appears to behave as a quasispecies. Assessment of original patient samples is preferable for understanding clinical SMSV populations.

Characterization of an RNA-Cleaving Deoxyribozyme with Optimal Activity at pH 5

An in vitro selection endeavor previously executed by our laboratory led to the isolation of a set of RNA-cleaving deoxyribozymes that thrive under acidic conditions [Liu, Z., Mei, S. H., Brennan, J. D., and Li, Y. (2003) J. Am. Chem. Soc. 125, 7539-7545]. One of these sequences, coined pH5DZ1, is a 100-nucleotide (nt) cis-acting enzyme that was found to exhibit high cleavage activity near pH 5. Herein, we seek to deduce the properties and sequence requirements of this enzyme. This deoxyribozyme was found to cleave a 23-nt chimeric DNA-RNA substrate, which contains a single ribonucleotide flanked by fluorophore- and quencher-modified nucleotides on each side of the cleavage junction. Extensive nucleotide deletion experiments indicated that only 42 bases within the original enzyme sequence are required for catalysis. Results from a reselection experiment further revealed that 26 of these nucleotides are absolutely conserved. In addition to sequence analysis and minimization studies, we successfully designed a trans-acting variant of this enzyme. Characterization of the cleavage products produced upon pH5DZ1-mediated RNA cleavage and analyses of possible structures of pH5DZ1 provided us with insights into the catalytic mechanism of pH5DZ1 and characteristics of deoxyribozymes that retain their activity under acidic conditions.

Comparative analysis of the large fragment of the 5′ untranslated region (LF-5′ UTR) of serotype A foot-and-mouth disease virus field isolates from India

India is endemic for foot-and-mouth disease (FMD) and in recent years a unique group within serotype A, carrying a codon deletion at an antigenically critical site in capsid protein VP3 has emerged (VP3(59)-deletion group). This tempted us to analyze the noncoding region, which is an under represented area, though critically associated with virus biology and pathogenesis. Analysis of the large fragment of 5' untranslated region (LF-5' UTR) of type A FMD virus revealed discrepancy in the overall tree topology between LF-5' UTR and 1D region possibly due to independent evolution of coding and noncoding regions. The VP3(59)-deletion group maintained its phylogenetic distinctness even at the LF-5' UTR. Eighteen lineage specific signatures detected here support independent evolutionary paths for the lineages. Extensive deletions of 45 and 89 nucleotides corresponding to the pseudoknot region were noticed. Conservation pattern in the 'A(253)AACA' motif in the cre/bus stem-loop indicates the importance of first three 'A' residues in VPg uridylylation. Of the three polypyrimidine tract binding protein (PTB) binding sites mapped on the internal ribosome entry site (IRES), the pyrimidine tract (Py tract) in the loop of domain 2 was found to be maximally conserved and it might be the major PTB binding site. Strikingly, a deletion group lineage specific transversion was noticed in the Py tract at the 3' end of IRES without significantly affecting its in vitro infectious titer. Hence, we presume that for efficient cap-independent viral translation, either a minimum number of pyrimidine residues rather than a complete Py tract or a Py tract tolerating transversions only at specific locations and a core motif 'CUUU' within the Py tract is essential.

Uncoupling Human Immunodeficiency Virus Type 1 gag and pol Reading Frames: Role of the Transframe Protein p6* in Viral Replication

Apart from its regulatory role in protease (PR) activation, little is known about the function of the human immunodeficiency virus type 1 transframe protein p6* in the virus life cycle. p6* is located between the nucleocapsid and PR domains in the Gag-Pol polyprotein precursor and is cleaved by PR during viral maturation. We have recently reported that the central region of p6* can be extensively mutated without abolishing viral infectivity and replication in vitro. However, mutagenesis of the entire p6*-coding sequence in the proviral context is not feasible without affecting the superimposed frameshift signal or the overlapping p1-p6(gag) sequences. To overcome these limitations, we created a novel NL4-3-derived provirus by displacing the original frameshift signal to the 3' end of the gag gene, thereby uncoupling the p6* gene sequence from the p1-p6(gag) reading frame. The resulting virus (AL) proved to be replication competent in different cell cultures and thus represents an elegant tool for detailed analysis of p6* function. Hence, extensive deletions or substitutions were introduced into the p6* gene sequence of the AL provirus, and effects on particle release, protein processing, and viral infectivity were evaluated. Interestingly, neither the deletion of 63% of all p6* residues nor the partial substitution by a heterologous sequence affected virus growth and infectivity, suggesting that p6* is widely dispensable for viral in vitro replication. However, the insertion of a larger reporter sequence interfered with virus production and maturation, implying that the length or conformation of this spacer region might be critical for p6* function.

Molecular characterization of toxigenic and atoxigenicAspergillus flavusisolates, collected from peanut fields in China

The objectives of this study were to assess the genetic relationships between toxigenic and atoxigenic isolates of Aspergillus flavus collected from peanut fields in China, and to analyse deletions within the aflatoxin biosynthetic gene cluster for the atoxigenic isolates. Analysis of random-amplified polymorphic DNA and microsatellite-primed PCR data showed that the toxigenic and atoxigenic isolates of A. flavus were not clustered based on their regions and their ability of aflatoxin and sclerotial production. These results were further supported by DNA sequence of ITS, pksA and omtA genes. PCR assays showed that 24 of 35 isolates containing no detectable aflatoxins had the entire aflatoxin gene cluster. Eleven atoxigenic isolates had five different deletion patterns in the cluster. Toxigenic and atoxigenic isolates of A. flavus are genetically similar, but some atoxigenic isolates having deletions within the aflatoxin gene cluster can be identified readily by PCR assays. Because the extensive deletions within the aflatoxin gene cluster are not rare in the atoxigenic isolates, analysis of deletion within the cluster would be an effective method for the rapid screening of atoxigenic isolates for developing biocontrol agents.

T‐cell tolerance induced by repeated antigen stimulation: Selective loss of Foxp3− conventional CD4 T cells and induction of CD4 T‐cell anergy

Repeated immunization of mice with bacterial superantigens induces extensive deletion and anergy of reactive CD4 T cells. Here we report that the in vitro proliferation anergy of CD4 T cells from TCR transgenic mice immunized three times with staphylococcal enterotoxin B (SEB) (3 x SEB) is partially due to an increased frequency of Foxp3(+) CD4 T cells. Importantly, reduced number of conventional CD25(-) Foxp3(-) cells, rather than conversion of such cells to Foxp3(+) cells, was the cause of that increase and was also seen in mice repeatedly immunized with OVA (3 x OVA) and OVA-peptide (OVAp) (3 x OVAp). Cell-transfer experiments revealed profound but transient anergy of CD4 T cells isolated from 3 x OVAp and 3x SEB mice. However, the in vivo anergy was CD4 T-cell autonomous and independent of Foxp3(+) Treg. Finally, proliferation of transferred CD4 T cells was inhibited in repeatedly immunized mice but inhibition was lost when transfer was delayed, despite the maintenance of elevated frequency of Foxp3(+) cells. These data provide important implications for Foxp3(+) cell-mediated tolerance in situations of repeated antigen exposure such as human persistent infections.

Lipid Binding to the Tail Domain of Vinculin

Vinculin is a highly conserved and abundant cytoskeletal protein involved in linking the actin cytoskeleton to the cell membrane at sites of cellular adhesion. At these sites of adhesion, vinculin plays a role in physiological processes such as cell motility, migration, development, and wound healing. Loss of normal vinculin function has been associated with cancer phenotypes, cardiovascular disease, and lethal errors in embryogenesis. The tail domain of vinculin (Vt) binds to acidic phospholipids and has been proposed to play a role in vinculin activation and focal adhesion turnover. To better characterize Vt-lipid specificity, we conducted a series of lipid co-sedimentation experiments and find that Vt shows specific association with phosphatidylinositol 4,5-bisphosphate (PIP2), compared with phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), or phosphatidylinositol (PI) in the context of mixed lipid vesicles. The C terminus of Vt has been proposed to be important for PIP2 association, as various mutations and deletions within the C-terminal reduce PIP2 association. Lipid co-sedimentation and NMR analyses indicate that removal of the hydrophobic hairpin does not alter Vt structure or PIP2 association. However, more extensive deletions within the C-terminal introduce Vt structural perturbations and reduce PIP2 binding. Intriguingly, a significant increase in PIP2 binding was observed for multiple Vt variants that perturb interactions between the N-terminal strap and helix bundle, suggesting that a rearrangement of this N-terminal strap may be required for PIP2 binding.