Alpha-satellite Sequences Research Articles

Enhancement of DNA hypomethylation alterations by gastric and bile acids promotes chromosomal instability in Barrett’s epithelial cell line

Gastric and bile acid reflux leads to chronic inflammation, resulting in methylation alterations in Barrett’s esophagus (BE) together with chromosomal instability (CIN). We investigated DNA hypomethylation following acid exposure and confirmed its significance in BE-related carcinogenesis by inducing CIN in vitro. OACP4C, an esophageal cancer cell line, and CP-A, a non-dysplastic cell line originating from BE, were exposed to acidic conditions using deoxycholic acid. CP-A exhibited substantially increased DNA hypomethylation of alpha satellite sequences in the centromere region, as well as increased levels of alpha satellite transcripts, but no changes were observed in the long interspersed nucleotide element-1 sequences distributed throughout the entire genome. These changes were not clearly found in OACP4C. Copy number changes at specific chromosomes were identified in CP-A, along with an increased number of cells exhibiting abnormal segregations, whereas these changes were rarely observed in OACP4C. The changes were maintained after several cell divisions. These findings suggest that alpha satellites are likely targets of DNA hypomethylation induced by acid exposure. CP-A was more sensitive to acid exposure than OACP4C, indicating that acid-induced DNA hypomethylation is involved in cancer development rather than progression, which could be involved in the underlying mechanism of esophagogastric junction carcinoma development.

Testing for ROS1, ALK, MET, and HER2 rearrangements and amplifications in a large series of biliary tract adenocarcinomas.

Biliary tract carcinomas are divided into intrahepatic, perihilar, distal extrahepatic cholangiocarcinomas, and gallbladder adenocarcinomas. Therapies targeting ROS1, ALK, MET, and HER2 alterations are currently evaluated in clinical trials. We assessed ROS1 and ALK translocations/amplifications as well as MET and HER2 amplifications for each tumor subtype by fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) in 73 intrahepatic, 40 perihilar bile duct, 36 distal extrahepatic cholangiocarcinomas, and 45 gallbladder adenocarcinomas (n = 194). By FISH, we detected targetable alterations in 5.2% of cases (n = 10): HER2 and MET amplifications were found in 4.1% (n = 8) and 1.0% (n = 2), respectively. The HER2-amplified cases were mostly gallbladder adenocarcinomas (n = 5). The MET- and HER2-amplified cases were all positive by IHC. Fourteen cases without MET amplification were positive by IHC, whereas HER2 over-expression was detected by IHC only in HER2-amplified cases. We detected no ALK or ROS1 translocation or amplification. Several alterations were consistent with aneuploidy: 24 cases showed only one copy of ROS1 gene, 4 cases displayed a profile of chromosomal instability, and an over-representation of centromeric alpha-satellite sequences was found in five cases. We confirm a relatively high rate of HER2 amplifications in gallbladder adenocarcinomas and the efficacy of IHC to screen these cases. Our results also suggest the value of IHC to screen MET amplification. Contrary to initial publications, ROS1 rearrangements seem to be very rare in biliary tract adenocarcinomas. We confirm a relatively high frequency of aneuploidy and chromosomal instability and reveal the over-representation of centromeric alpha-satellite sequences in intrahepatic cholangiocarcinomas.

Effect of electromagnetic field exposure on the transcription of repetitive DNA elements in human cells

ABSTRACTRepetitive DNA (RE-DNA) was long thought to be silent and inert; only recent research has shown that it can be transcribed and that transcription alteration can be induced by environmental stress conditions, causing human pathological effects. The aim of this study was to determine whether exposure to radiofrequency electromagnetic fields (RF-EMF) could affect the transcription of RE-DNA. To this purpose, three different human cell lines (HeLa, BE(2)C and SH-SY5Y) were exposed to 900 MHz GSM-modulated RF-EMF at specific absorption rate of 1 W/kg or to sham. After exposure, mRNA levels of RE-DNA were evaluated through quantitative real-time PCR. The following RE-DNA types were investigated: Long Interspersed nucleotide Element 1, DNA alpha satellite and Human Endogenous Retroviruses-like sequences. When comparing cells exposed to RF-EMF versus control samples, different results were found for the three cell lines evaluated, indicating that RF-EMF exposure can significantly affect RE-DNA transcription and that the effects strongly depend on the cellular context and the tissue type. Further studies are needed to elucidate which molecular mechanisms could be involved.

The meiotic segregation pattern of a reciprocal translocation t(10;12)(q26.1;p13.3) by fluorescence in situ hybridization sperm analysis.

The meiotic segregation of chromosomes 10 and 12 was analyzed in a male heterozygous for a reciprocal translocation, t(10;12)(q26.1;p13.3), using fluorescence in situ hybridization (FISH). Centromeric specific probes that detect alpha satellite sequences of chromosomes 10 and 12 were used. A total of 10,049 spermatozoa were analyzed. The frequencies of alternate/adjacent 1, adjacent 2, and 3:1 modes of segregation were: 84.25, 10.95%, and 4.42%, respectively. Diploidy was present in 0.23% of spermatozoa. Similar segregation patterns have been reported for this donor by direct karyotyping of sperm cells. FISH is a valuable technique for studying meiotic segregation patterns in that larger samples can be studied in a relatively short time. However, it does not provide information on the full chromosome complement of the spermatozoon.

Molecular characterization of ageratum enation virus and DNA-satellites associated with yellowing and leaf curl symptoms on mulberry in Pakistan

The whitefly-transmitted begomoviruses (family Geminiviridae) infect dicotyledonous plants and occur in tropical and sub-tropical regions. Although most commonly found in herbaceous plants, recently begomoviruses have increasingly been identified in woody plants. Leaf samples from five mulberry (Morus alba L.) plants with leaf yellowing and curling symptoms were collected in Lahore (Pakistan) and shown by PCR to be associated with a begomovirus, an alphasatellite and a betasatellite. The complete sequences of two begomovirus clones, as well as an alphasatellite clone and a betasatellite clone, were determined. The begomovirus clones were shown to be isolates of ageratum enation virus (AEV), a virus most commonly identified in weeds but increasingly being identified in crops such as tomato, soybean and fenugreek. Analyses of the sequences of the alphasatellite and betasatellite clones showed them to be isolates of Guar leaf curl alphasatellite and Papaya leaf curl betasatellite, respectively. Both the virus and the alphasatellite sequences showed evidence of a recombination. This is the first report of the weed-infecting monopartite begomovirus AEV, and associated satellites, infecting the woody plant mulberry.

Two distinct begomoviruses associated with an alphasatellite coinfecting Emilia sonchifolia in Thailand.

Emilia sonchifolia is a traditionally used medicinal plant that is widespread in tropical and subtropical regions of the world. Yellow vein symptoms were observed in E. sonchifolia plants in fields in the county of Koh Samui, Surat Thani Province, Thailand, in August 2015. Two distinct begomoviruses, designated TH4872-6 and TH4872-9, and an associated alphasatellite were obtained from an E. sonchifolia leaf sample (TH4872). Sequence analysis showed that the full-length sequence of TH4872-6 was most closely related to that of ageratum yellow vein China virus (AYVCNV), with 85.7% identity, suggesting that it is a novel begomovirus, while the TH4872-9 sequence closely resembled cotton leaf curl Multan virus (CLCuMuV) with 99.1% identity. The alphasatellite sequence showed the highest nucleotide sequence identity (92.8%) to an isolate of tobacco curly shoot alphasatellite (TbCSA) originating from China. Recombination analysis revealed that the isolate TH4872-6 is a potential recombinant begomovirus, derived from ageratum yellow vein virus (AYVV) and tobacco leaf curl Thailand virus (TbLCTHV). This study represents the first report of begomoviruses identified in E. sonchifolia in Thailand.

The survival of transplanted mesenchymal stem cells from humans Wharton’s jelly of the umbilical cord in the central nervous system of rats with experimental allergic encephalomyelitis after their suboccipital injection

Human Wharton’s Jelly of mesenchymal stem cells (hWJ-MSCs) have a considerable advantage in comparison with the cells from other sources; their therapeutic potential in treating the central nervous system (CNS) diseases is higher than that of other MSCs. That’s why hWJ-MSCs can be a new alternative treatment of CNS demyelinization damages including multiple sclerosis (MS).Purpose. To study the persistence and distribution of hWJ-MSCs in different CNS segments following suboccipital transplantation into cerebrospinal fluid (CSF) of rats with experimental allergic encephalomyelitis (EAE).Methods. Isolation and cultivation of hWJ-MSCs in vitro. Immunological phenotyping by flow cytometry. EAE induction. Suboccipital injection of MSCs into EAE rats cerebrospinal fluid. Persistence of hWJ-MSCs in the CNS of EAE rats was assayed by PCR in tissue samples at days 2, 3, 4 and 5 using primers for amplifying nucleic alpha satellite sequences of human 17th chromosome.Results. PCR-assays for alpha-satellite sequences revealed human DNA to be detected in the treated rats within 5 days after suboccipital injection at the peak of disease. The human DNA was traced in CSF and various segments of the spinal cord.Conclusions. The data obtained suggest that suboccipitally delivered hWJ-MSCs, survive and can migrate through the CSF from the injection site (cisterna magna) to various segments of CNS.

Molecular characterization of a new begomovirus infecting Mirabilis jalapa in northern India.

Begomoviruses are whitefly-transmitted single-stranded DNA viruses that are responsible for considerable economic losses. A begomovirus, alphasatellite and betasatellite were characterized in a Mirabilis jalapa plant exhibiting severe leaf curling and mottling symptoms. The complete viral genome shared highest sequence identity of 87% with pedilanthus leaf curl virus (AM712436), reported from Pakistan. Additionally, the viral genome was 84% identical to that of chilli leaf curl India virus (KX951415) and 83% identical to that of tobacco curly shoot virus (GU1999584), which were previously reported to infect M. jalapa in India and China, respectively. Based on the ICTV criterion for begomovirus species demarcation (≥91% sequence identity for the complete genome), the virus represents a new species, for which we propose the name Mirabilis leaf curl virus. The alphasatellite and betasatellite sequences were similar to the corresponding sequences of ageratum yellow vein India alphasatellite (KU852743; 99% identity) and tomato leaf curl Patna betasatellite (HQ180394; 86% identity) sequences, respectively. This report describes a new begomovirus-satellite disease complex in M. jalapa.

Clusters of alpha satellite on human chromosome 21 are dispersed far onto the short arm and lack ancient layers.

Human alpha satellite (AS) sequence domains that currently function as centromeres are typically flanked by layers of evolutionarily older AS that presumably represent the remnants of earlier primate centromeres. Studies on several human chromosomes reveal that these older AS arrays are arranged in an age gradient, with the oldest arrays farthest from the functional centromere and arrays progressively closer to the centromere being progressively younger. The organization of AS on human chromosome 21 (HC21) has not been well-characterized. We have used newly available HC21 sequence data and an HC21p YAC map to determine the size, organization, and location of the AS arrays, and compared them to AS arrays found on other chromosomes. We find that the majority of the HC21 AS sequences are present on the p-arm of the chromosome and are organized into at least five distinct isolated clusters which are distributed over a larger distance from the functional centromere than that typically seen for AS on other chromosomes. Using both phylogenetic and L1 element age estimations, we found that all of the HC21 AS clusters outside the functional centromere are of a similar relatively recent evolutionary origin. HC21 contains none of the ancient AS layers associated with early primate evolution which is present on other chromosomes, possibly due to the fact that the p-arm of HC21 and the other acrocentric chromosomes underwent substantial reorganization about 20 million years ago.

First Report of Okra enation leaf curl virus and Associated Cotton leaf curl Multan betasatellite and Cotton leaf curl Multan alphasatellite Infecting Cotton in Pakistan: A New Member of the Cotton Leaf Curl Disease Complex.

Cotton (Gossypium hirsutum L.) is an important and widely cultivated crop in Pakistan, upon which many rely for economic security. Cotton leaf curl disease (CLCuD) is caused by a complex comprising of more than eight species in the genus Begomovirus (family Geminiviridae) with associated betasatellite and alphasatellites. During 2011, characteristic symptoms of leaf curl disease were widespread (>40%), and the whitefly Bemisia tabaci (Genn.) vector of the leaf curl complex was abundant in commercial cotton fields in Burewala, Pakistan. Symptoms included vein thickening, upward or downward leaf curling, and foliar enations. To test for the presence of a begomovirus(es), total DNA was extracted from 100 mg of symptomatic leaf tissues from five different plants (isolates CLCuDBur1 to 5) using the CTAB method (1). Total DNA extracts were used for rolling circle amplification (RCA) using TempliPhi DNA Amplification Kit (GE Healthcare). Of the five field isolates, the RCA product for only one, CLCuDBur3, digested with HindIII, produced an apparently full-length ~2.7 kb fragment, suggesting that CLCuD-Bur3 represented a distinct isolate. The 2.7-kb fragment was cloned into the plasmid vector pGEM-3Zf+ (Promega, Madison, WI). To test for the presence of associated alphasatellites and betasatellites, the PCR primers, AlphaF/R and BetaF/R (2), were used to amplify the putative 1.4-kbp molecules. The resultant 1.4-kb PCR products were ligated into the pGEMT-Easy vector and cloned. Cloned inserts for each were subjected to DNA sequencing, bidirectionally. The cloned monopartite, helper begomovirus genome (HF567945), one betasatellite (HF567946), and one alphasatellite (HF567947) sequences were determined and found to be 2,742, 1,358, and 1,376 bases long, respectively. Pairwise sequence comparisons were carried out for each using the 10 most closely related species or strains (identified in GenBank using BLASTn) using MEGA5 software. The CLCuDBur3 genome sequence shared its highest identity (99.6%) with Okra enation leaf curl virus (OELCuV) (KC019308), so CLCuDBur3 is a variant of OELCuV, a begomovirus reported previously from Abelmoschus esculentus (L.) (okra) plants in India. The betasatellite and alphasatellite shared their highest nt identity at 96 and 98.7% with Cotton leaf curl Multan betasatellite (CLCuMB) (AM774311) and Cotton leaf curl Multan alphasatellite (CLCuMA), respectively (misnamed as CLCuBuA in GenBank) (FN658728). Additionally, the HindIII-digested RCA products were analyzed by Southern blot hybridization using a DIG-labeled DNA probe specific for the intergenic region of either Cotton leaf curl Burewala virus (CLCuBuV) or OELCuV. The OELCuV, but not the CLCuBuV, probe hybridized with HindIII digested RCA products (CLCuDBur3 genome), confirming the presence of OELCuV and the absence of CLCuBuV, the latter being the most prevalent begomovirus species infecting cotton in Pakistan. This is the first report of OELCuV infecting cotton plants in Pakistan, underscoring the discovery of yet another begomovirus member of the CLCuD complex. Further, the possible co-infection of cotton by OELCuV and other recognized species of the CLCuD complex could facilitate further diversification (potentially, through recombination) and lead to the emergence of new variants with the potential to cause damage to the cotton crop in Pakistan.

Replication of alpha-satellite DNA arrays in endogenous human centromeric regions and in human artificial chromosome.

In human chromosomes, centromeric regions comprise megabase-size arrays of 171 bp alpha-satellite DNA monomers. The large distances spanned by these arrays preclude their replication from external sites and imply that the repetitive monomers contain replication origins. However, replication within these arrays has not previously been profiled and the role of alpha-satellite DNA in initiation of DNA replication has not yet been demonstrated. Here, replication of alpha-satellite DNA in endogenous human centromeric regions and in de novo formed Human Artificial Chromosome (HAC) was analyzed. We showed that alpha-satellite monomers could function as origins of DNA replication and that replication of alphoid arrays organized into centrochromatin occurred earlier than those organized into heterochromatin. The distribution of inter-origin distances within centromeric alphoid arrays was comparable to the distribution of inter-origin distances on randomly selected non-centromeric chromosomal regions. Depletion of CENP-B, a kinetochore protein that binds directly to a 17 bp CENP-B box motif common to alpha-satellite DNA, resulted in enrichment of alpha-satellite sequences for proteins of the ORC complex, suggesting that CENP-B may have a role in regulating the replication of centromeric regions. Mapping of replication initiation sites in the HAC revealed that replication preferentially initiated in transcriptionally active regions.

The Past, Present, and Future of Human Centromere Genomics

The centromere is the chromosomal locus essential for chromosome inheritance and genome stability. Human centromeres are located at repetitive alpha satellite DNA arrays that compose approximately 5% of the genome. Contiguous alpha satellite DNA sequence is absent from the assembled reference genome, limiting current understanding of centromere organization and function. Here, we review the progress in centromere genomics spanning the discovery of the sequence to its molecular characterization and the work done during the Human Genome Project era to elucidate alpha satellite structure and sequence variation. We discuss exciting recent advances in alpha satellite sequence assembly that have provided important insight into the abundance and complex organization of this sequence on human chromosomes. In light of these new findings, we offer perspectives for future studies of human centromere assembly and function.

A naturally occurring defective DNA satellite associated with a monopartite begomovirus: evidence for recombination between alphasatellite and betasatellite.

Monopartite begomoviruses and their associated satellites form unique disease complexes that have emerged as a serious threat to agriculture worldwide. It is well known that frequent recombination contributes to the diversification and evolution of geminiviruses. In this study, we identified a novel defective satellite molecule (RecSat) in association with Tobacco leaf curl Yunnan virus (TbLCYNV) in a naturally infected tobacco plant. Sequence analysis showed that Recsat comprises 754 nucleotides in size and is a chimera involving alphasatellite and betasatellite sequences, containing both betasatellite-conserved region and alphasatellite stem-loop structure. Recombination analysis revealed that RecSat has arisen from three independent recombination events likely involving Tomato yellow leaf curl China betasatellite, Ageratum yellow vein China betasatellite and Tobacco curly shoot alphasatellite. Co-inoculation of RecSat with TbLCYNV induced symptoms indistinguishable from those induced by TbLCYNV alone in Nicotiana benthamiana. Southern blot hybridization showed that RecSat could be trans-replicated stably in N. benthamiana plants by TbLCYNV, and impaired the accumulation of helper virus and co-inoculated alphasatellite. Our results provide the first evidence for recombination between two distinct types of satellites among geminivirus complex and highlight recombination as a driving force for geminivirus evolution.

First Detection of Cotton leaf curl Burewala virus and Cognate Cotton leaf curl Multan betasatellite and Gossypium darwinii symptomless alphasatellite in Symptomatic Luffa cylindrica in Pakistan.

Cotton leaf curl disease (CLCuD) is the major plant viral constraint to cotton production on the Indian subcontinent (2). CLCuD is primarily caused by begomovirus, Cotton leaf curl Burewala virus (CLCuBuV), and Cotton leaf curl Multan betasatellite (CLCuMB). During 2011 in Burewala, Pakistan, plants in a production field of Luffa cylindrica (Ghia tori) were infested with the whitefly Bemisia tabaci (Genn.), and ~60% of the plants exhibited leaf curling and stunting symptoms, reminiscent of those caused by begomoviruses (Geminiviridae). Total DNA was extracted from five different symptomatic leaf samples using the CTAB method (1), and extracts were analyzed by Southern blot hybridization. As a probe, we used a 1.1-kbp fragment of CLCuBuV and a positive signal was obtained from all five samples. Total DNA was used as template for rolling circle amplification (RCA) using the TempliPhi DNA Amplification Kit (GE Healthcare, Little Chalfont, United Kingdom). The amplified RCA products were digested with EcoRI, and the resulting ~2.7-kbp fragments from each isolate were directionally cloned into the EcoRI digested, pGEM-3Zf+ (Promega, Madison, WI) plasmid vector. PCR was used to amplify the prospective, associated betasatellite and alphasatellite molecules using the primers BetaF5'-GGTACCGCCGGAGCTTAGCWCKCC-3' and BetaR5'-GGTACCGTAGCTAAGGCTGCTGCG-3', and AlphaF5'-AAGCTTAGAGGAAACTAGGGTTTC-3' and AlphaR5'-AAGCTTTTCATACARTARTCNCRDG-3', respectively. The putative satellite amplicons, at ~1.4 kbp each were cloned in the plasmid vector pGEMT-Easy (Promega, Madison, WI) and sequenced. BLASTn comparisons of the apparently full-length begomoviral genomes, at 2,753 nt, against the NCBI database revealed that all five isolates were most closely related to CLCuBuV (FR750321). In addition, one each of beta- and alpha-satellite were amplified from all five samples at 1,393 and 1,378 bases, respectively. The beta- and alpha-satellites were most closely related to CLCuMB (HE985228) and the Gossypium darwinii symptomless alphasatellite (GDaSA) (FR877533), respectively. Pairwise sequence comparisons of the top 10 BLASTn hits using MEGA5 indicated that the helper begomovirus shared 99.9% identity with CLCuBuV (FR750321), the most prevalent helper virus currently associated with the leaf curl complex in Pakistan. Based on the ICTV demarcation for begomoviral species at <89%, it is considered a variant of CLCuBuV. The resultant beta- and alpha-satellite sequences were 98.1% and 97.8% identical to CLCuMB (HE985228) and GDaSA (FR877533), respectively, and are the most prevalent satellites associated with the CLCuD complex in Pakistan and India (2). To our knowledge, this is first report of the CLCuBuV-CLCuMB-GDaSA complex infecting a cucurbitaceous species, and the first report of L. cylindrica as a host of the CLCuD complex. This discovery of CLCuBuV and associated satellites in a cucurbitaceous host that is widely grown in Pakistan and India where this complex infects cotton indicates that the host range of CLCuBuV is broader than expected. This new information will aid in better understanding of cotton leaf curl disease epidemiology in the current epidemic areas.

Sequences Associated with Centromere Competency in the Human Genome

Centromeres, the sites of spindle attachment during mitosis and meiosis, are located in specific positions in the human genome, normally coincident with diverse subsets of alpha satellite DNA. While there is strong evidence supporting the association of some subfamilies of alpha satellite with centromere function, the basis for establishing whether a given alpha satellite sequence is or is not designated a functional centromere is unknown, and attempts to understand the role of particular sequence features in establishing centromere identity have been limited by the near identity and repetitive nature of satellite sequences. Utilizing a broadly applicable experimental approach to test sequence competency for centromere specification, we have carried out a genomic and epigenetic functional analysis of endogenous human centromere sequences available in the current human genome assembly. The data support a model in which functionally competent sequences confer an opportunity for centromere specification, integrating genomic and epigenetic signals and promoting the concept of context-dependent centromere inheritance.

A newly characterized human well-differentiated liposarcoma cell line contains amplifications of the 12q12-21 and 10p11-14 regions

While surgery is the usual treatment for localized well-differentiated and dedifferentiated liposarcomas (WDLPS/DDLPS), the therapeutic options for patients with advanced disease are limited. The classical antimitotic treatments are most often inefficient. The establishment of genetically characterized cell lines is therefore crucial for providing in vitro models for novel targeted therapies. We have used spectral karyotyping, fluorescence in situ hybridization with whole chromosome painting and locus-specific probes, and array-comparative genomic hybridization to identify the chromosomal and molecular alterations of a novel cell line established from a recurring sclerosing WDLPS. The karyotype was hypertriploid and showed multiple structural anomalies. All cells retained the presence of a giant marker chromosome that had been previously identified in the primary cell cultures. This giant chromosome contained high-level amplification of chromosomal regions 12q13-21 and lacked the alpha-satellite centromeric sequences associated with WDLPS/DDLPS. The 12q amplicon was large, containing 370 amplified genes. The DNA copy number ranged from 3 to 57. The highest levels of amplification were observed at 12q14.3 for GNS, WIF1, and HMGA2. We analyzed the mRNA expression status by real-time reverse transcription polymerase chain reaction for six genes from this amplicon: MDM2, HMGA2, CDK4, TSPAN31, WIF1, and YEATS4. mRNA overexpression was correlated with genomic amplification. A second amplicon originating from 10p11-14 was also present in the giant marker chromosome. The 10p amplicon contained 62 genes, including oncogenes such as MLLT10, previously described in chimeric fusion with MLL in leukemias, NEBL, and BMI1.

Global Repeat Map Method for Higher Order Repeat Alpha Satellites in Human and Chimpanzee Genomes (Build 37.2 Assembly)

Alpha satellites are tandemly repeated sequences found in all human centromeres. In addition to the functional and structural role within centromere they are also a suitable model for evolutionary studies, because of being subject to concerted evolution. The Global Repeat Map (GRM) algorithm is a convenient computational tool to determine consensus repeat units and their exact size within a given genomic sequence, both of monomeric and higher-order (HOR) type. Using GRM, we identify in Build 37.2 assembly fifteen different alpha satellite HORs, three of them novel, not reported previously. In the next step we compute suprachromosomal family classification and CENP-B box / pJα distributions for these HORs. All human alpha satellite sequences originate from one pra-ancestral alpha satellite monomer. For the first time we perform GRM analysis and compare human and chimpanzee alpha satellite HORs for chromosomes 4 and give an evidence that the human and chimpanzee alpha satellites originate from a common ancestor that predated the human-chimpanzee separation. We also compare the codon-like trinucleotide (CLT) extensions of human and chimpanzee chromosome 4. Our results are consistent with the expectation that the alpha satellite HORs in human and chimpanzee have been created after the human- chimpanzee separation.

Isolation and Characterization of the Etheostoma tallapoosae (Teleostei: Percidae) CENP-A Gene

Both centromeric alpha-satellite sequences as well as centromeric protein A (CENP-A) are highly variable in eukaryotes. CENP-A, a histone H3 variant, is thought to act as the epigenetic “mark” for assembly of centromeric proteins. While most of the histone fold domain (HFD) of the CENP-A is fairly well conserved, a portion of this HFD as well as the N-terminal tail show adaptive variation in both plants and animals. Such variation may establish reproductive barriers that may lead to speciation. The family Percidae contains over 200 species most of which are within the subfamily Etheostomatinae. This subfamily represents a species rich radiation of freshwater fishes in North America and these species exhibit both allopatric and sympatric distributions. In order to study the evolution of CENP-A in percid fish species, we have isolated and characterized the CENP-A gene from Etheostoma tallapoosae by PCR based gene walking. As a result of this study we have demonstrated that the Tallapoosa darter CENP-A gene HFD sequences can be isolated from genomic DNA by nested PCR in a manner that does not lead to the amplification of the highly sequence related histone H3 gene. We also demonstrated that PCR based walking can be subsequently used to isolate the rest of the CENP-A gene and adjacent gene sequences. These adjacent gene sequences provide us with a primer binding sites for PCR isolation of the CENP-A gene from other percid species of fishes. An initial comparison of three percid species shows that the N-terminal tail of the percid CENP-A gene shows adaptive evolution.

Ageratum conyzoides: A host to a unique begomovirus disease complex in Cameroon

Ageratum conyzoides (goat weed) is a widespread uncultivated species in Cameroon that exhibits leaf curl disease (LCD) symptoms suggestive of begomovirus infection. In Asia, different begomovirus–satellite complexes have been identified in A. conyzoides. The objective of this study was to determine the identity of the suspect begomoviruses and their associated satellites in A. conyzoides in Cameroon. The results indicated that all three symptomatic A. conyzoides plants examined were infected with a new begomovirus species, herein named Ageratum leaf curl Cameroon virus (ALCCMV). The ALCCMV genome sequences shared their highest identity, at 84.3–88.5%, with a group of tomato-infecting begomoviruses from West Africa. In addition, a betasatellite and an alphasatellite were cloned from the same symptomatic A. conyzoides plants. The betasatellite sequences shared limited sequence identity at 37% or less with the betasatellite Cotton leaf curl Gezira betasatellite, and the new betasatellite species is herein named Ageratum leaf curl Cameroon betasatellite (ALCCMB). The alphasatellite shared 80% nt identity with Tomato leaf curl Cameroon alphasatellite (ToLCCMA), and the new alphasatellite species is herein named Ageratum leaf curl Cameroon alphasatellite (ALCCMA). In addition, two fragments containing begomovirus–alphasatellite sequences were cloned from sample AGLI4, and they were related to the defecting interfering molecule (Y14167) associated with Ageratum yellow vein virus from Asia. These results suggest that the begomoviral–satellite complexes infecting A. conyzoides in Cameroon may be as complex or more so, to species and strains reported thus far from Asia.

533 SUBTYPING OF RENAL CELL TUMORS USING A MULTICOLOUR FLUORESCENCE IN SITU HYBRIDIZATION BASED ASSAY

You have accessJournal of UrologyKidney Cancer: Evaluation and Staging1 Apr 2011533 SUBTYPING OF RENAL CELL TUMORS USING A MULTICOLOUR FLUORESCENCE IN SITU HYBRIDIZATION BASED ASSAY Jimsgene Sanjmyatav, Britta Meyer, Heiko Wunderlich, Mieczyslaw Gajda, Marc-Oliver Grimm, and Kerstin Junker Jimsgene SanjmyatavJimsgene Sanjmyatav Jena, Germany More articles by this author , Britta MeyerBritta Meyer Bremerhaven, Germany More articles by this author , Heiko WunderlichHeiko Wunderlich Jena, Germany More articles by this author , Mieczyslaw GajdaMieczyslaw Gajda Jena, Germany More articles by this author , Marc-Oliver GrimmMarc-Oliver Grimm Jena, Germany More articles by this author , and Kerstin JunkerKerstin Junker Jena, Germany More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1257AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES An accurate identification of kidney tumor entities based on histopathological features alone is in some cases difficult. Accordingly a genetic analysis of tumors could be helpful for an exact diagnosis of kidney tumors. To date there are no genetic tests available for the precise subtyping of these tumors. We developed a genetic subtyping multicolour fluorescence in situ hybridization (FISH) assay based on common copy number aberrations of the most frequent renal cell tumors. METHODS For this study we used a combined FISH assay containing region specific probe for VHL gene labelled in green, red labelled probe targeting region 1p12, gold labelled alpha satellite sequences of chromosome 7 and blue-labelled probe which target alpha satellite region of chromosome 17. Overall 61 kidney tumors were analyzed by FISH containing 26 clear cell renal cell carcinomas (ccRCC), 10 papillary (pRCC), 13 chromophobe (chRCC) and 12 benign oncocytomas. As reference to FISH analysis we carried out array-CGH for all analyzed tumors. RESULTS The correlation of FISH findings with histopathological data revealed that histological subtypes were correct in 100% (26/26) of ccRCC cases, in 80% (8/10) of pRCC, 100% (13/13) in chRCC and in 33% (4/12) of oncocytomas. Two tumors classified as papillary according to histopathological features were grouped to ccRCC on the basis of genetic pattern detected by FISH and CGH analysis. In 8 oncocytomas we found either no genomic alterations or alterations in less than 10% of scored nuclei. Array-CGH and CGH analysis demonstrated no alterations or atypical chromosomal changes in these tumors. CONCLUSIONS The current study demonstrates that the developed multicolor FISH assay allows an accurate identification of the most frequent renal cell tumor subtypes and increases the diagnostic accuracy of RCC. It represents a robust and rapid method for routine diagnostics in cases of uncertain diagnosis by histopathological evaluation and helps to overcome the limitations of pathological classification. Currently we are working on a second set of probe composed of centromeric probes of chromosomes 2 und 6 and a translocation probe for chromosome 11 allowing the precise differentiation of some ambiguous cases of chromophobe RCC and oncocytomas as well. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e216-e217 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jimsgene Sanjmyatav Jena, Germany More articles by this author Britta Meyer Bremerhaven, Germany More articles by this author Heiko Wunderlich Jena, Germany More articles by this author Mieczyslaw Gajda Jena, Germany More articles by this author Marc-Oliver Grimm Jena, Germany More articles by this author Kerstin Junker Jena, Germany More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...