Evidence of multiple transcription initiation and termination sites within the rDNA intergenic spacer and rRNA readthrough transcription in the urochordate Herdmania curvata

Analysis of the structure of the urochordate Herdmania curvata ribosomal DNA intergenic spacer (IGS) and its role in transcription initiation and termination suggests that rRNA gene regulation in this chordate differs from that in vertebrates. A cloned H, curvata IGS is 1881 bp and composed predominantly of two classes of similar repeat sequences that largely alternate in a tandem array. Southern blot hybridization demonstrates that the IGS length variation within an individual and population is largely the result of changes in internal repeat number. Nuclease S1 mapping and primer extension analyses suggest that there are two transcription initiation sites at the 3' end of the most 3' repetitive element; these sites are 6 nucleotides apart. Unlike mouse, Xenopus, and Drosophila, there is no evidence of transcription starting elsewhere in the IGS. Most sequence differences between the promoter repeat and the other internal repeats are in the vicinity of the putative initiation sites. As in Drosophila, nuclease S1 mapping of transcription termination sites suggest that there is not a definitive stop site and a majority of the pre-rRNAs read through a substantial portion of the IGS. Some transcription appears to proceed completely through the promoter repeat into the adjacent rDNA unit. Analysis of oocyte RNA by reverse transcription-polymerase chain reaction (RT-PCR) confirms that readthrough transcription into the adjacent rDNA unit is occurring in some small IGS length variants; there is no evidence of complete readthrough of IGSs larger than 1.0 kb.

The GHEP-EMPOP collaboration on mtDNA population data-A new resource for forensic casework

Mitochondrial DNA (mtDNA) population data for forensic purposes are still scarce for some populations, which may limit the evaluation of forensic evidence especially when the rarity of a haplotype needs to be determined in a database search. In order to improve the collection of mtDNA lineages from the Iberian and South American subcontinents, we here report the results of a collaborative study involving nine laboratories from the Spanish and Portuguese Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG) and EMPOP. The individual laboratories contributed population data that were generated throughout the past 10 years, but in the majority of cases have not been made available to the scientific community. A total of 1019 haplotypes from Iberia (Basque Country, 2 general Spanish populations, 2 North and 1 Central Portugal populations), and Latin America (3 populations from Sao Paulo) were collected, reviewed and harmonized according to defined EMPOP criteria. The majority of data ambiguities that were found during the reviewing process (41 in total) were transcription errors confirming that the documentation process is still the most error-prone stage in reporting mtDNA population data, especially when performed manually. This GHEP-EMPOP collaboration has significantly improved the quality of the individual mtDNA datasets and adds mtDNA population data as valuable resource to the EMPOP database (www.empop.org). (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Direct Interrogation of Viral Peptides Presented by the Class I HLA of HIV-Infected T Cells

Identification of CD8+ cytotoxic T lymphocyte (CTL) epitopes has traditionally relied upon testing of overlapping peptide libraries for their reactivity with T cells in vitro. Here, we pursued deep ligand sequencing (DLS) as an alternative method of directly identifying those ligands that are epitopes presented to CTLs by the class I human leukocyte antigens (HLA) of infected cells. Soluble class I HLA-A*11:01 (sHLA) was gathered from HIV-1 NL4-3-infected human CD4+ SUP-T1 cells. HLA-A*11:01 harvested from infected cells was immunoaffinity purified and acid boiled to release heavy and light chains from peptide ligands that were then recovered by size-exclusion filtration. The ligands were first fractionated by high-pH high-pressure liquid chromatography and then subjected to separation by nano-liquid chromatography (nano-LC)–mass spectrometry (MS) at low pH. Approximately 10 million ions were selected for sequencing by tandem mass spectrometry (MS/MS). HLA-A*11:01 ligand sequences were determined with PEAKS software and confirmed by comparison to spectra generated from synthetic peptides. DLS identified 42 viral ligands presented by HLA-A*11:01, and 37 of these were previously undetected. These data demonstrate that (i) HIV-1 Gag and Nef are extensively sampled, (ii) ligand length variants are prevalent, particularly within Gag and Nef hot spots where ligand sequences overlap, (iii) noncanonical ligands are T cell reactive, and (iv) HIV-1 ligands are derived from de novo synthesis rather than endocytic sampling. Next-generation immunotherapies must factor these nascent HIV-1 ligand length variants and the finding that CTL-reactive epitopes may be absent during infection of CD4+ T cells into strategies designed to enhance T cell immunity.

Chloroplast DNA footprints of postglacial recolonization by oaks

Recolonization of Europe by forest tree species after the last glaciation is well documented in the fossil pollen record. This spread may have been achieved at low densities by rare events of long-distance dispersal, rather than by a compact wave of advance, generating a patchy genetic structure through founder effects. In long-lived oak species, this structure could still be discernible by using maternally transmitted genetic markers. To test this hypothesis, a fine-scale study of chloroplast DNA (cpDNA) variability of two sympatric oak species was carried out in western France. The distributions of six cpDNA length variants were analyzed at 188 localities over a 200 × 300 km area. A cpDNA map was obtained by applying geostatistics methods to the complete data set. Patches of several hundred square kilometers exist which are virtually fixed for a single haplotype for both oak species. This local systematic interspecific sharing of the maternal genome strongly suggests that long-distance seed dispersal events followed by interspecific exchanges were involved at the time of colonization, about 10,000 years ago.

Efficient loading of HLA-DR with a T helper epitope by genetic exchange of CLIP

The HLA class II-associated invariant chain (Ii)-derived peptide (CLIP) occupies the peptide binding groove during assembly in the endoplasmic reticulum, travels with HLA class II to endosomal compartments, and is subsequently released to allow binding of antigenic peptides. We investigated whether the exchange of CLIP with a known T helper epitope at the DNA level would lead to efficient loading of this helper epitope onto HLA class II. For this purpose, a versatile Ii-encoding expression vector was created in which CLIP can be replaced with a helper epitope of choice. Upon supertransfection of HLA-DR1-transfected 293 cells with an Ii vector encoding a known T helper epitope (HA307–319), predominantly length variants of this epitope were detected in association with the HLA-DR1 molecules of these cells. Moreover, this transfectant was efficiently recognized by a peptide-specific T helper clone (HA1.7). The results suggest that this type of Ii vector can be used to create potent class II+ cellular vaccines in which defined T cell epitopes are continuously synthesized.

Polymorphic simple sequence repeat regions in chloroplast genomes: applications to the population genetics of pines.

Simple sequence repeats (SSRs), consisting of tandemly repeated multiple copies of mono-, di-, tri-, or tetranucleotide motifs, are ubiquitous in eukaryotic genomes and are frequently used as genetic markers, taking advantage of their length polymorphism. We have examined the polymorphism of such sequences in the chloroplast genomes of plants, by using a PCR-based assay. GenBank searches identified the presence of several (dA)n.(dT)n mononucleotide stretches in chloroplast genomes. A chloroplast (cp) SSR was identified in three pine species (Pinus contorta, Pinus sylvestris, and Pinus thunbergii) 312 bp upstream of the psbA gene. DNA amplification of this repeated region from 11 pine species identified nine length variants. The polymorphic amplified fragments were isolated and the DNA sequence was determined, confirming that the length polymorphism was caused by variation in the length of the repeated region. In the pines, the chloroplast genome is transmitted through pollen and this PCR assay may be used to monitor gene flow in this genus. Analysis of 305 individuals from seven populations of Pinus leucodermis Ant. revealed the presence of four variants with intrapopulational diversities ranging from 0.000 to 0.629 and an average of 0.320. Restriction fragment length polymorphism analysis of cpDNA on the same populations previously failed to detect any variation. Population subdivision based on cpSSR was higher (Gst = 0.22, where Gst is coefficient of gene differentiation) than that revealed in a previous isozyme study (Gst = 0.05). We anticipate that SSR loci within the chloroplast genome should provide a highly informative assay for the analysis of the genetic structure of plant populations.

Long Terminal Repeat Sequence Analysis of HTLV-2 Molecular Variants Identified in Southern Brazil

In Brazil, human T-lymphotropic virus type 2 (HTLV-2) is endemic in Amerindians and epidemic in intravenous drug users (IDUs). The long terminal repeat (LTR) is the most divergent genomic region of HTLV-2, therefore useful to characterize subtypes. Nucleotide sequence and restriction fragment length polymorphism (RFLP) analysis of LTR genomic segments of fourteen HTLV-2 strains isolated from HIV-infected patients of Londrina, Southern Brazil, were carried out. Molecular analysis disclosed that all HTLV-2 strains belonged to 2a subtype, and RFLP detected the presence of the a4, a5, and a6 subgroups according to Switzer's nomenclature. RFLP correlated with nucleotide sequence, and phylogenetic analysis clustered HTLV-2 sequences of IDUs into subgroups a5 and a6. HTLV-2 sequences from individuals of sexual risk factor clustered into the a4 subgroup. These results extend the knowledge of the genetic diversity of HTLV-2 circulating in Brazil and provide insights into HTLV-2 transmission and virus movement in this geographic area.

Risk of multiple myeloma is associated with polymorphisms within telomerase genes and telomere length

Compelling biological and epidemiological evidences point to a key role of genetic variants of the TERT and TERC genes in cancer development. We analyzed the genetic variability of these two gene regions using samples of 2,267 multiple myeloma (MM) cases and 2,796 healthy controls. We found that a TERT variant, rs2242652, is associated with reduced MM susceptibility (OR?=?0.81; 95% CI: 0.72-0.92; p?=?0.001). In addition we measured the leukocyte telomere length (LTL) in a subgroup of 140 cases who were chemotherapy-free at the time of blood donation and 468 controls, and found that MM patients had longer telomeres compared to controls (OR?=?1.19; 95% CI: 0.63-2.24; ptrend ?=?0.01 comparing the quartile with the longest LTL versus the shortest LTL). Our data suggest the hypothesis of decreased disease risk by genetic variants that reduce the efficiency of the telomerase complex. This reduced efficiency leads to shorter telomere ends, which in turn may also be a marker of decreased MM risk.

Combinatorial effects of splice variants modulate function of Aiolos

The transcription factor Aiolos (also known as IKZF3), a member of the Ikaros family of zinc-finger proteins, plays an important role in the control of B lymphocyte differentiation and proliferation. Previously, multiple isoforms of Ikaros family members arising from differential splicing have been described and we now report a number of novel isoforms of Aiolos. It has been demonstrated that full-length Ikaros family isoforms localize to heterochromatin and that they can associate with complexes containing histone deacetylase (HDAC). In this study, for the first time we directly investigate the cellular localization of various Aiolos isoforms, their ability to heterodimerize with Ikaros and associate with HDAC-containing complexes, and the effects on histone modification and binding to putative targets. Our work demonstrates that the cellular activities of Aiolos isoforms are dependent on combinations of various functional domains arising from the differential splicing of mRNA transcripts. These data support the general principle that the function of an individual protein is modulated through alternative splicing, and highlight a number of potential implications for Aiolos in normal and aberrant lymphocyte function.

Inactivation of L-type calcium channels is determined by the length of the N terminus of mutant beta(1) subunits.

Voltage-dependent calcium channel (Ca(v)) pores are modulated by cytosolic beta subunits. Four beta-subunit genes and their splice variants offer a wide structural array for tissue- or disease-specific biophysical gating phenotypes. For instance, the length of the N terminus of beta(2) subunits has major effects on activation and inactivation rates. We tested whether a similar mechanism principally operates in a beta(1) subunit. Wild-type beta(1a) subunit (N terminus length 60 aa) and its newly generated N-terminal deletion mutants (51, 27 and 18 aa) were examined within recombinant L-type calcium channel complexes (Ca(v)1.2 and alpha(2)delta2) in HEK293 cells at the whole-cell and single-channel level. Whole-cell currents were enhanced by co-transfection of the full-length beta(1a) subunit and by all truncated constructs. Voltage dependence of steady-state activation and inactivation did not depend on N terminus length, but inactivation rate was diminished by N terminus truncation. This was confirmed at the single-channel level, using ensemble average currents. Additionally, gating properties were estimated by Markov modeling. In confirmation of the descriptive analysis, inactivation rate, but none of the other transition rates, was reduced by shortening of the beta(1a) subunit N terminus. Our study shows that the length-dependent mechanism of modulating inactivation kinetics of beta(2) calcium channel subunits can be confirmed and extended to the beta(1) calcium channel subunit.

Independent evolution of hypervariable regions of HIV-1 gp120: V4 as a swarm of N-Linked glycosylation variants

In this study we have characterized intra-patient length polymorphism in V4 by cloning and sequencing a C2-C4 fragment from HIV plasma RNA in patients at different stages of HIV disease. Clonal analysis of clade B, G, and CRF02 isolates during early infection shows extensive intra-patient V4 variability, due to the presence of indel-associated polymorphism. Indels, coupled to amino acid substitution events, affect the number and distribution of potential N-glycosylation sites, resulting in the coexistence, within the same patient, of V4 subsets, each characterized by different sizes, amino acid sequences, and potential N-glycosylation patterns. In contrast, V3 appears to be relatively homogeneous, with similar V3 associated to significantly different V4 within the same clinical specimen. Based on these data, we propose that during early chronic infection V4 is present as a highly divergent quasispecies, enabling the virus to adopt different conformational structures according to immune constrains and other selective pressures

Linkage disequilibrium between a melanin-based colour polymorphism and tail length in the barn owl

A linkage disequilibrium between sexually selected and life history traits can be explained by three mutually non-exclusive mechanisms. Genes coding for two traits may be located close on the same chromosome, genes responsible for variation in one of the trait may pleiotropically alter the other, and non-random pairing with respect to two traits may generate a non-physical linkage disequilibrium between their genes. Knowledge of which of these three mechanisms is responsible for a covariation between two traits is of interest to understand why differently ornamented individuals differ in several phenotypic aspects. In Switzerland, barn owls Tyto alba mate randomly with respect to a colour polymorphism generating a large range of variants between reddish-brown and white, males being lighter coloured than females. Several studies have shown that plumage coloration is not neutral with respect to some life history components. To test whether coloration is genetically associated with body size, partial cross-fostering experiments were performed by exchanging some hatchlings between nests. These experiments showed that darker biological fathers produce longer-tailed offspring. This sex-specific pattern is consistent with the hypothesis of non-physical linkage disequilibrium. In line with this hypothesis, darker coloured males were mated with longer-tailed females, whereas female coloration was not associated with tail length of their mate. The finding that dark nestlings had a longer tail than their pale siblings also supports the physical linkage and pleiotropy hypotheses. Therefore, non-random pairing can generate or strengthen a genetic covariation between a secondary sexual character and a morphological trait.

Breast implant-associated anaplastic large cell lymphoma: two distinct clinicopathological variants with different outcomes.

BACKGROUND: ALK-negative anaplastic large cell lymphoma associated with breast implant (i-ALCL) has been recently recognized as a distinct entity. Among 43 830 lymphomas registered in the French Lymphopath network since 2010, 300 breast lymphomas comprising 25 peripheral T-cell lymphomas (PTCL) were reviewed. Among PTCL, ALK-negative ALCL was the most frequent and all of them were associated with breast implants. PATIENTS AND METHODS: Since 2010, all i-ALCL cases were collected from different institutions through Lymphopath. Immuno-morphologic features, molecular data and clinical outcome of 19 i-ALCLs have been retrospectively analyzed. RESULTS: The median age of the patients was 61 years and the median length between breast implant and i-ALCL was 9 years. Most implants were silicone-filled and textured. Implant removal was performed in 17 out of 19 patients with additional treatment based on mostly CHOP or CHOP-like chemotherapy regimens (n = 10/19) or irradiation (n = 1/19). CHOP alone or ABVD following radiation without implant removal have been given in two patients. The two clinical presentations, i.e. effusion and less frequently tumor mass correlated with distinct histopathologic features: in situ i-ALCL (anaplastic cell proliferation confined to the fibrous capsule) and infiltrative i-ALCL (pleomorphic cells massively infiltrating adjacent tissue with eosinophils and sometimes Reed-Sternberg-like cells mimicking Hodgkin lymphoma). Malignant cells were CD30-positive, showed a variable staining for EMA and were ALK negative. Most cases had a cytotoxic T-cell immunophenotype with variable T-cell antigen loss and pSTAT3 nuclear expression. T-cell receptor genes were clonally rearranged in 13 out of 13 tested cases. After 18 months of median follow-up, the 2-year overall survival for in situ and infiltrative i-ALCL was 100% and 52.5%, respectively. CONCLUSIONS: In situ i-ALCLs have an indolent clinical course and generally remain free of disease after implant removal. However, infiltrative i-ALCLs could have a more aggressive clinical course that might require additional therapy to implant removal.

Phenotypic and proteomic characterization of multiply antibiotic-resistant variants of Salmonella entetica serovar typhimurium selected following exposure to disinfectants

In previous work, Salmonella enterica serovar Typhimurium strain SL1344 was exposed to sublethal concentrations of three widely used farm disinfectants in daily serial passages for 7 days in an attempt to investigate possible links between the use of disinfectants and antimicrobial resistance. Stable variants OXCR1, QACFGR2, and TOPR2 were obtained following treatment with an oxidizing compound blend, a quaternary ammonium disinfectant containing formaldehyde and glutaraldehyde, and a tar acid-based disinfectant, respectively. All variants exhibited ca. fourfold-reduced susceptibility to ciprofloxacin, chloramphenicol, tetracycline, and ampicillin. This coincided with reduced levels of outer membrane proteins for all strains and high levels of AcrAB-To1C for OXCR1 and QACFGR2, as demonstrated by two-dimensional high-performance liquid chromatography-mass spectrometry. The protein profiles of OXCR1 and QACFGR2 were similar, but they were different from that of TOPR2. An array of different proteins protecting against oxidants, nitroaromatics, disulfides, and peroxides were overexpressed in all strains. The growth and motility of variants were reduced compared to the growth and motility of the parent strain, the expression of several virulence proteins was altered, and the invasiveness in an enteric epithelial cell line was reduced. The colony morphology of OXCR1 and QACFGR2 was smooth, and both variants exhibited a loss of modal distribution of the lipopolysaccharide O-antigen chain length, favoring the production of short O-antigen chain molecules. Metabolic changes were also detected, suggesting that there was increased protein synthesis and a shift from oxidative phosphorylation to substrate level phosphorylation. In this study, we obtained evidence that farm disinfectants can select for strains with reduced susceptibility to antibiotics, and here we describe changes in protein expression in such strains.

Piezotolerant small-colony variants with increased thermotolerance, antibiotic susceptibility, and low invasiveness in a clonal Staphylococcus aureus population

Following a pressure treatment of a clonal Staphylococcus aureus culture with 400 MPa for 30 min, piezotolerant variants were isolated. Among 21 randomly selected survivors, 9 were piezotolerant and all formed small colonies on several agar media. The majority of the isolates showed increased thermotolerance, impaired growth, and reduced antibiotic resistance compared to the wild type. However, several nonpiezotolerant isolates also demonstrated impaired growth and the small-colony phenotype. In agglutination tests for the detection of protein A and fibrinogen, the piezotolerant variants showed weaker agglutination reactions than the wild type and the other isolates. All variants also showed defective production of the typical S. aureus golden color, a characteristic which has previously been linked with virulence. They were also less able to invade intestinal epithelial cells than the wild type. These S. aureus variants showed phenotypic similarities to previously isolated Listeria monocytogenes piezotolerant mutants that contained mutations in ctsR. Because of these similarities, possible alterations in the ctsR hypermutable regions of the S. aureus variants were investigated through amplified fragment length polymorphism analysis. No mutations were identified, and subsequently we sequenced the ctsR and hrcA genes of three representative variants, finding no mutations. This work demonstrates that S. aureus probably possesses a strategy resulting in an abundance of multiple-stressresistant variants within clonal populations. This strategy, however, seems to involve genes and regulatory mechanisms different from those previously reported for L. monocytogenes. We are in the process of identifying these mechanisms.