Telomeric Ends Of Chromosomes Research Articles

Interaction analysis of anti-cancer drug Methotrexate with bcl-2 promoter stabilization and its transcription regulation

G-quadruplex (G4) motifs are higher-order secondary structures putatively present in the promoter region of several oncogenes and telomeric ends of chromosomes. The stabilization of G-quadruplex structures in the promoters of proto-oncogenes using specific small molecules provides a prominent strategy for the development of anti-cancer therapeutics. Methotrexate is an FDA-approved anti-cancer drug that is known to inhibit dihydrofolate reductase (DHFR) activity that leads to cell death. Here, in this study, we employed a broad range of bio-physical techniques to understand the interaction of Methotrexate with G-quadruplex motifs present in the proto-oncogenes (bcl-2, c-myc, c-kit21) and telomeric region (tel22). Interestingly, Methotrexate showed the highest binding affinity and specificity for bcl-2 G4 (Kd1 = 9 nM) as compared to other G-quadruplex, and duplex DNA. Circular dichroism melting analysis depicted higher stabilization of bcl-2 G4 by the Methotrexate. While molecular docking and dynamic simulation analysis revealed stacking mode of interaction with the G4 structure. Furthermore, in a cellular-based assay, Methotrexate demonstrated higher toxicity against A549 lung cancer cells over other cancer cells such as PC-3, HeLa, and HepG2. Quantitative RT-PCR and western blot results provide direct evidence that Methotrexate treatment to A549 lung cancer cells could downregulate the transcription and translation of the bcl-2 with the stabilization of G-quadruplex motif. In summary, this report provides valuable information about the alternative molecular mechanism of Methotrexate for the treatment of cancer and offers new insight into its anti-cancer activity.

Interchromosomal Transfer of Immune Regulation During Infection of Barley with the Powdery Mildew Pathogen.

Powdery mildew pathogens colonize over 9500 plant species, causing critical yield loss. The Ascomycete fungus, Blumeria graminis f. sp. hordei (Bgh), causes powdery mildew disease in barley (Hordeum vulgare L.). Successful infection begins with penetration of host epidermal cells, culminating in haustorial feeding structures, facilitating delivery of fungal effectors to the plant and exchange of nutrients from host to pathogen. We used expression Quantitative Trait Locus (eQTL) analysis to dissect the temporal control of immunity-associated gene expression in a doubled haploid barley population challenged with Bgh. Two highly significant regions possessing trans eQTL were identified near the telomeric ends of chromosomes (Chr) 2HL and 1HS. Within these regions reside diverse resistance loci derived from barley landrace H. laevigatum (MlLa) and H. vulgare cv. Algerian (Mla1), which associate with the altered expression of 961 and 3296 genes during fungal penetration of the host and haustorial development, respectively. Regulatory control of transcript levels for 299 of the 961 genes is reprioritized from MlLa on 2HL to Mla1 on 1HS as infection progresses, with 292 of the 299 alternating the allele responsible for higher expression, including Adaptin Protein-2 subunit μ AP2M and Vesicle Associated Membrane Protein VAMP72 subfamily members VAMP721/722. AP2M mediates effector-triggered immunity (ETI) via endocytosis of plasma membrane receptor components. VAMP721/722 and SNAP33 form a Soluble N-ethylmaleimide-sensitive factor Attachment Protein REceptor (SNARE) complex with SYP121 (PEN1), which is engaged in pathogen associated molecular pattern (PAMP)-triggered immunity via exocytosis. We postulate that genes regulated by alternate chromosomal positions are repurposed as part of a conserved immune complex to respond to different pathogen attack scenarios.

Observation of Extensive Chromosome Axis Remodeling during the "Diffuse-Phase" of Meiosis in Large Genome Cereals.

The production of balanced fertile haploid gametes requires the faithful separation of paired (synapsed) chromosomes toward the end of meiotic prophase I (desynapsis). This involves the timely dissolution of the synaptonemal complex during the pachytene-diplotene transition, a stage traditionally referred to as the “diffuse stage.” In species with large genomes such as, barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) we know most about the early stages of meiotic prophase I. There, synapsis initiates at the telomeric ends of chromosomes and progresses toward the centromeric regions through the ordered assembly of the synaptonemal complex (SC). Synapsis is impacted by recombination (crossing over, CO) which locally modifies the extent of chromatin compaction and extension. CO is uneven along the chromosomes, occurring mainly toward the telomeric regions resulting in a highly skewed distribution of recombination events. However, we know very little about the process of desynapsis which occurs during the “diffuse stage,” where the synapsed and recombined chromosomes faithfully desynapse and separate into daughter cells. Here, using 3D-SIM super-resolution immuno-cytology combined with the use of antibodies directed against two crucial SC proteins, ASY1 and ZYP1, we followed the whole of meiosis I (i.e., both synapsis and desynapsis) in both barley and wheat. We showed that synapsis forms a characteristic tri-partite SC structure in zygotene (more clearly seen in barley). Toward the end of meiosis I, as the SC starts to disassemble, we show that extensive chromosome axis remodeling results in the formation of characteristic “tinsel-like” structures in both wheat and barley. By using a mutant (des10) that is severely compromised in polymerization of ZYP1during synapsis, we show that tinsel structure formation during SC dissolution is not dependant on full synapsis and may relate instead to changes in expansion stress. Our observations highlight a potentially new role for ASYNAPSIS1 (ASY1) in desynapsis, in addition to chromosome synapsis and cohesion.

Abstract 266: The G-quadruplex ligand EMICORON potentiates the antitumor efficacy of chemotherapy on colon cancer experimental models

Abstract Background. G-quadruplex (G4) structures, present at the telomeric ends of chromosomes and in the promoters of a wide range of genes important in cell signaling, gained interest as therapeutic targets and several small molecules able to bind and stabilize G4 structures have been developed. However, due to the poor drug-like properties and/or selectivity profile none of the G4 ligands has progressed through the drug discovery pipeline. Our group recently showed that the novel G4 ligand EMICORON exhibited a favourable pharmacological profile, was well tolerated in mice and elicited antitumor efficacy against advanced experimental models of colon cancer. Based on these results, our aim was to study the ability of EMICORON to increase the efficacy of the standard chemotherapy for human colon cancer. Matherials and Methods. We assessed the in vitro cytotoxic activity of EMICORON in combination with SN-38, 5-Fluorouracil and Oxaliplatin by clonogenic and 3D tumor-spheroid formation assay on HT29 colon cancer cells. The combination index (CI) values indicating synergism (CI 0.9 1.2) was evaluated. In vivo experiments were performed in mice bearing HT29 tumors treated with Irinotecan followed by EMICORON in one or two cycles of administration. Moreover, efficacy of the standard FOLFIRI (5-Fluorouracil, Leucovorin and Irinotecan) or FOLFOX (5-Fluorouracil, Leucovorin and Oxaliplatin) was evaluated in two different colon cancer patient-derived xenografts (PDXs). Results and conclusions. The exposure of HT29 cells to the combination of EMICORON with chemotherapeutics resulted strongly synergistic when the drugs were administered following the sequence SN-38→EMICORON, EMICORON→5-Fluoruracil or EMICORON→Oxaliplatin, while the opposite sequences were additive or slightly antagonistic. The high activity of SN-38 followed by EMICORON was confirmed in the HT29-formed spheroids. When mice bearing HT29 xenografts were treated with EMICORON or Irinotecan, a tumor weight inhibition (TWI) of 35 or 65% was observed respectively, while the treatment with Irinotecan followed by EMICORON resulted more effective with a TWI of 80%. This marked antitumor effect of the combination produced an increase in overall survival of mice of 85%. Interestingly, the administration of a second cycle of treatment produced an impressive increase of survival of mice to 114%. Then, we tested the efficacy of FOLFOX or FOLFIRI therapeutic regimens on two colon cancer PDXs and as expected these treatments were very effective in reducing the tumor mass and delaying the tumor regrowth, but the relapse of the disease was observed in all the mice. In conclusion, our results demonstrating that EMICORON is able to increase the potency of any single drug, provide a compelling argument to suggest that the integration of EMICORON in standard chemotherapeutic regimens could be a highly valuable strategy. Citation Format: Manuela Porru, Simona Artuso, Luca Pompili, Carla Caruso, Armandodoriano Bianco, Marcella Mottolese, Carla A. Amoreo, Annamaria Biroccio, Carlo Leonetti. The G-quadruplex ligand EMICORON potentiates the antitumor efficacy of chemotherapy on colon cancer experimental models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 266.

Modeling of Plasmodium falciparum Telomerase Reverse Transcriptase Ternary Complex: Repurposing of Nucleoside Analog Inhibitors.

The Plasmodium falciparum telomerase reverse transcriptase (PfTERT) is a ribonucleoprotein that assists the maintenance of the telomeric ends of chromosomes by reverse transcription of its own RNA subunit. It represents an attractive therapeutic target for eradication of the plasmodial parasite at the asexual liver stage. Automated modeling using MUSTER and knowledge-based techniques were used to obtain a three-dimensional model of the active site of reverse transcriptase domain of PfTERT, which is responsible for catalyzing the addition of incoming dNTPs to the growing DNA strand in presence of divalent magnesium ions. Further, the ternary complex of the active site of PfTERT bound to a DNA-RNA duplex was also modeled using Haddock server and represents the functional form of the enzyme. Initially, established nucleoside analog inhibitors of PfTERT, AZTTP, and ddGTP were docked in the modeled binding site of the PfTERT ternary complex using AutoDock v4.2. Subsequently, docking studies were carried out with 14 approved nucleoside analog inhibitors. Docking studies predicted that floxuridine, gemcitabine, stavudine, and vidarabine have high affinity for the PfTERT ternary complex. Further analysis on the basis of known side effects led us to propose repositioning of vidarabine as a suitable drug candidate for inhibition of PfTERT.

Stability and free energy calculation of LNA modified quadruplex: a molecular dynamics study

Telomeric ends of chromosomes, which comprise noncoding repeat sequences of guanine-rich DNA, which are the fundamental in protecting the cell from recombination and degradation. Telomeric DNA sequences can form four stranded quadruplex structures, which are involved in the structure of telomere ends. The formation and stabilization of telomeric quadruplexes has been shown to inhibit the activity of telomerase, thus establishing telomeric DNA quadrulex as an attractive target for cancer therapeutic intervention. Molecular dynamic simulation offers the prospects of detailed description of the dynamical structure with ion and water at molecular level. In this work we have taken a oligomeric part of human telomeric DNA, d(TAGGGT) to form different monomeric quadruplex structures d(TAGGGT)₄. Here we report the relative stabilities of these structures under K⁺ ion conditions and binding interaction between the strands, as determined by molecular dynamic simulations followed by energy calculation. We have taken locked nucleic acid (LNA) in this study. The free energy molecular mechanics Poission Boltzman surface area calculations are performed for the determination of most stable complex structure between all modified structures. We calculated binding free energy for the combination of different strands as the ligand and receptor for all structures. The energetic study shows that, a mixed hybrid type quadruplex conformation in which two parallel strands are bind with other two antiparallel strands, are more stable than other conformations. The possible mechanism for the inhibition of the cancerous growth has been discussed. Such studies may be helpful for the rational drug designing.

138. GENOME-WIDE LINKAGE SCAN FOR FAMILIAL DIZYGOTIC TWINNING

The tendency to conceive dizygotic (DZ) twins is a complex trait influenced by genetic and environmental factors. To search for new candidate loci for twinning we have conducted a genome-wide linkage scan in 525 families using microsatellite and single nucleotide polymorphism (SNP) marker panels. Non-parametric linkage analyses including 523 families containing a total of 1115 mothers of DZ twins (MODZT) from Australia and New Zealand (ANZ) and The Netherlands (NL) produced four linkage peaks above the threshold for suggestive linkage, including a highly suggestive peak at the extreme telomeric end of chromosome 6 with an exponential (exp)LOD score of 2.813 (P = 0.0002). Since the DZ twinning rate increases steeply with maternal age independent of genetic effects, we also investigated linkage including only families where at least one MODZT gave birth to her first set of twins before the age of 30. These analyses produced a maximum expLOD score of 2.718 (p = 0.0002), largely due to linkage signal from the ANZ cohort, however, ordered subset analyses indicated this result is most likely a chance finding in the combined dataset. Linkage analyses were also performed for two large DZ twinning families from the USA, one of which produced a peak on chromosome 2 in the region of two potential candidate genes. Sequencing of FSHR and FIGLA, along with INHBB in MODZTs from two large NL families with family-specific linkage peaks directly over this gene, revealed a potentially functional variant in the 5’ untranslated region of FSHR that segregated with the DZ twinning phenotype in the UT family. Work is continuing screening candidate genes. Our data provide further evidence for complex inheritance of familial DZ twinning.

Genome-wide linkage scan localizes the harlequin locus in the Great Dane to chromosome 9

Harlequin is a coat pattern of the Great Dane characterized by ragged patches of full color on a white background. Harlequin patterning is a bigenic trait, resulting from the interaction of the merle allele of SILV, and a dominant modifier locus, H. Breeding data suggest that H is embryonic recessive lethal and that all harlequins are Hh. To identify linkage with the harlequin phenotype, 46 Great Danes from 5 pedigrees were genotyped for 280 microsatellite markers in a whole genome screen. One marker on the telomeric end of chromosome 9 was suggestive of linkage. Fine mapping of this region using additional microsatellite markers and 10 Great Danes from a sixth pedigree resulted in significant LOD scores for 2 markers. Reported herein is linkage mapping of the H locus to a 3.27 Mb region of chromosome 9 containing approximately 20 genes.

Genetic dissection of Sle1c (130.16)

Abstract The Sle1c locus, derived from the NZW mouse strain, is a ~6.5Mb interval located at the telomeric end of chromosome 1. Previous work has shown that when Sle1c is congenically bred onto a B6 background to yield B6. Sle1cw, a mild lupus-like disease occurs in aged mice (>8mo.), indicated by low to moderate levels of anti-nuclear antibodies (ANA) and low incidence of glomerulonephritis (GN). Further phenotypic analyses of this strain reveal decreased T-dependant (TD) immune responses, abnormal germinal centers (GC), increased T cell activation and proliferation, amplification of chronic graft-versus-host disease (cGVHD), and decreased number of regulatory T cells (Tregs). Cr2 is located on the telomeric end of Sle1c. It encodes Complement receptor 1 (CR1) and 2 (CR2) thorough alternative splicing has previously been described as a candidate gene for Sle1c. It was shown with subcongenic strains of Sle1c that cr2w results in the abnormal GC and decreased TD immune response phenotypes. However, cr2w does not account for the other phenotypes. We have created a series of subcongenic strains on the centromeric end of Sle1c that map the T cell activation phenotype to <1Mb. Screening is ongoing for the enhanced cGVHD. This should lead to the identification of the corresponding gene(s). This research is supported by NIH R01 AI1045050

An update on sheep and goat linkage maps and other genomic resources

The linkage maps of sheep and goats are strikingly disparate in terms of comprehensiveness. Since its inception in 1995, the sheep linkage map has been regularly updated with the most recent version (4.6) released in June 2006. The sex averaged sheep linkage map has essentially complete coverage of the sheep genome, spans approximately 3580 cM, and is comprised of 1374 markers representing 1333 loci. The sheep is unusual among mammals in that the male autosomal map of 3817 cM is significantly longer than the female autosomal map of 3146 cM. About 50% of the male map expansion occurs at the centromeric and telomeric ends of chromosomes. In contrast to the situation for sheep, the most recent version of the goat linkage map, with the exception of the map for chromosome 1, was published in 1998 and is available only as a male map. The goat linkage map spans 2737 cM and is comprised of 307 loci, although many of the chromosomes contain two or more linkage groups. The sheep and goat linkage maps are primarily comprised of anonymous microsatellite markers, derived from sheep or cattle sequences. Most markers on the goat map have also been positioned on the sheep map, and many of the markers on the sheep and goat maps have also been positioned on the bovine linkage maps so the maps from all three species can be readily aligned. Several sheep and goat partial linkage maps have been developed as byproducts of QTL studies but many of these partial linkage maps are not publicly available. Several genomics resources are available for sheep and to a lesser degree, for goats, including genomic and cDNA libraries, a large collection of sheep EST sequences, sheep bacterial artificial chromosome (BAC) end sequences from a library with 12-fold genome coverage, mitochondrial sequence maps for sheep and goats, sheep microarrays, and web sites containing genomics and genetics information for both sheep and goats. In addition, two radiation hybrid panels are available for sheep, and a large-scale public single nucleotide polymorphism (SNP) project will lead to a 20 K sheep SNP chip and a haplotype map. Finally, a large number of trait mapping experiments have been or are currently being conducted in sheep and goats. The high degree of sequence and chromosome conservation between sheep, goats and cattle means that many of the cattle genomic resources will also be highly useful for sheep and goats.

Confirmation of quantitative trait loci for somatic cell score on bovine chromosome 18 in the German Holstein

Abstract. Fifty-one half-sib families with 2768 sires were selected for this study to confirm a quantitative trait locus (QTL) detected within a previous project initiated by the German Cattle Breeders Federation (ADR). The data based on a granddaughter design were divided into two parts and were analysed using linear models and paternal half sib regression methods. The results strongly support the hypothesis that the chromosomal region around marker TGLA227 at the telomeric end of chromosome 18 harbours a QTL for somatic cell score (SCS) in the German Holstein population. Using a two-QTL model the analysis showed evidence for another QTL for SCS in that region on chromosome 18. Further fine mapping studies should be carried out to decide on the two QTL hypothesis.

Discovery of quantitative trait loci for resistance to parasitic nematode infection in sheep: I. Analysis of outcross pedigrees

BackgroundCurrently most pastoral farmers rely on anthelmintic drenches to control gastrointestinal parasitic nematodes in sheep. Resistance to anthelmintics is rapidly increasing in nematode populations such that on some farms none of the drench families are now completely effective. It is well established that host resistance to nematode infection is a moderately heritable trait. This study was undertaken to identify regions of the genome, quantitative trait loci (QTL) that contain genes affecting resistance to parasitic nematodes.ResultsRams obtained from crossing nematode parasite resistant and susceptible selection lines were used to derive five large half-sib families comprising between 348 and 101 offspring per sire. Total offspring comprised 940 lambs. Extensive measurements for a range of parasite burden and immune function traits in all offspring allowed each lamb in each pedigree to be ranked for relative resistance to nematode parasites.Initially the 22 most resistant and 22 most susceptible progeny from each pedigree were used in a genome scan that used 203 microsatellite markers spread across all sheep autosomes. This study identified 9 chromosomes with regions showing sufficient linkage to warrant the genotyping of all offspring. After genotyping all offspring with markers covering Chromosomes 1, 3, 4, 5, 8, 12, 13, 22 and 23, the telomeric end of chromosome 8 was identified as having a significant QTL for parasite resistance as measured by the number of Trichostrongylus spp. adults in the abomasum and small intestine at the end of the second parasite challenge. Two further QTL for associated immune function traits of total serum IgE and T. colubiformis specific serum IgG, at the end of the second parasite challenge, were identified on chromosome 23.ConclusionDespite parasite resistance being a moderately heritable trait, this large study was able to identify only a single significant QTL associated with it. The QTL concerned adult parasite burdens at the end of the second parasite challenge when the lambs were approximately 6 months old. Our failure to discover more QTL suggests that most of the genes controlling this trait are of relatively small effect. The large number of suggestive QTL discovered (more than one per family per trait than would be expected by chance) also supports this conclusion.

Telomerase: Structure and properties of the enzyme, and peculiarities of yeast telomerase

Telomerase is a ribonucleoprotein that extends telomeric ends of chromosomes to counterbalance their natural shortening due to incomplete DNA replication in eukaryotic cells. The core enzyme consists of the catalytic reverse transcriptase subunit TERT (Telomerase Reverse Transcriptase) and the RNA subunit TER (Telomerase RNA), a short specific region of which serves as a template for synthesis of telomeric repeats. Despite the intensive research of telomerase in different organisms, the mechanism of its action remains unclear. This review focuses on telomerase of yeast Saccharomyces cerevisiae. Unlike ciliate and human telomerases, the yeast enzyme adds only one telomeric repeat to the DNA oligonucleotide (primer), imitating the single-stranded telomeric end of a chromosome, and remains stably bound to it after elongation in vitro. This review summarizes the results of numerous studies on the structure and functions of the core enzyme components, their interactions with each other and a primer, and telomerase activity on different substrates in vitro. The peculiarities of telomerase functioning in a cell and accessory proteins of the telomerase complex are also discussed.

Crystal structure of parallel quadruplexes from human telomeric DNA

Telomeric ends of chromosomes, which comprise noncoding repeat sequences of guanine-rich DNA, are fundamental in protecting the cell from recombination and degradation. Disruption of telomere maintenance leads to eventual cell death, which can be exploited for therapeutic intervention in cancer. Telomeric DNA sequences can form four-stranded (quadruplex) structures, which may be involved in the structure of telomere ends. Here we describe the crystal structure of a quadruplex formed from four consecutive human telomeric DNA repeats and grown at a K(+) concentration that approximates its intracellular concentration. K(+) ions are observed in the structure. The folding and appearance of the DNA in this intramolecular quadruplex is fundamentally different from the published Na(+)-containing quadruplex structures. All four DNA strands are parallel, with the three linking trinucleotide loops positioned on the exterior of the quadruplex core, in a propeller-like arrangement. The adenine in each TTA linking trinucleotide loop is swung back so that it intercalates between the two thymines. This DNA structure suggests a straightforward path for telomere folding and unfolding, as well as ways in which it can recognize telomere-associated proteins.

The Forty-First Midwinter Conference of Immunologists

Among NZB/W-derived New Zealand mixed (NZM) strains, only NZM/Aeg2410 (NZM2410) has been well characterized. In contrast to NZM2410, NZM2328 mice develop autoantibodies and acute and severe chronic glomerulonephritis (GN) with female predominance similarly to NZB/WF1 and humans with systemic lupus erythematosus (SLE). Chronic GN with glomerular sclerosis and tubular atrophy but not acute GN was correlated with severe proteinuria. In a backcross analysis of (NZM2328 X C57L/J) F1 X NZM2328, four SLE susceptibility genomic intervals were identified. One of them (Cgnz1) is on the telomeric end of chromosome 1 and close to Sle1. It was significantly linked to chronic GN. A locus (Agnz1) distinct from Cgnz1 on this interval was suggestively linked to acute GN. Two genetic intervals on chromosome 17 were also suggestively linked to acute GN, one of which is the H-2–Tnf complex, while the other (Agnz2) is on the distal end of the chromosome. A single locus (Adaz1) identified in the midregion of chromosome 4 in NZM2328 mice was suggestively linked to plasma levels of IgG anti-dsDNA autoantibodies. These results differ significantly from those in the backcross analysis of (NZM2410 X C57BL/6)F1 X NZM2410 by other investigators. They support the concept that different sets of genes are involved in acute and chronic GN. The genomic differences between the NZM strains and between C57L/J and C57BL/6 account for the differences between our analysis and that on NZM 2410. These results provide evidence for the importance of background genes on the expression of SLE, with implications for genetic studies of human SLE.

NZM2328: a new mouse model of systemic lupus erythematosus with unique genetic susceptibility loci.

Among NZB/W-derived New Zealand mixed (NZM) strains, only NZM/Aeg2410 (NZM2410) has been well characterized. In contrast to NZM2410, NZM2328 mice develop autoantibodies and acute and severe chronic glomerulonephritis (GN) with female predominance similarly to NZB/WF1 and humans with systemic lupus erythematosus (SLE). Chronic GN with glomerular sclerosis and tubular atrophy but not acute GN was correlated with severe proteinuria. In a backcross analysis of (NZM2328 X C57L/J) F1 X NZM2328, four SLE susceptibility genomic intervals were identified. One of them (Cgnz1) is on the telomeric end of chromosome 1 and close to Sle1. It was significantly linked to chronic GN. A locus (Agnz1) distinct from Cgnz1 on this interval was suggestively linked to acute GN. Two genetic intervals on chromosome 17 were also suggestively linked to acute GN, one of which is the H-2-Tnf complex, while the other (Agnz2) is on the distal end of the chromosome. A single locus (Adaz1) identified in the midregion of chromosome 4 in NZM2328 mice was suggestively linked to plasma levels of IgG anti-dsDNA autoantibodies. These results differ significantly from those in the backcross analysis of (NZM2410 X C57BL/6)F1 X NZM2410 by other investigators. They support the concept that different sets of genes are involved in acute and chronic GN. The genomic differences between the NZM strains and between C57L/J and C57BL/6 account for the differences between our analysis and that on NZM 2410. These results provide evidence for the importance of background genes on the expression of SLE, with implications for genetic studies of human SLE.

Identification of genetic loci associated with paralysis, inflammation and weight loss in mouse experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE), a model for human multiple sclerosis, is an inducible inflammatory and demyelinating disease of the central nervous system (CNS). Susceptibility to this disease is heritable and is demonstrated by the development of an ascending paralysis accompanied by a loss in body wt 2-3 weeks following immunization with proteins derived from CNS myelin. In a previous genetic analysis of susceptibility to EAE in a cross between susceptible SJL/J mice and resistant B10.S mice, we found suggestive evidence of linkage with disease susceptibility at the telomeric end of chromosome 2 and in the central region of chromosome 3. To define these associations more precisely and to investigate the genetic factors controlling measurable phenotypes of EAE, we performed a new analysis with a larger number of mice. The results now indicate that the chromosome 2 locus significantly influences EAE-related weight loss (P = 6.7 x 10(-5)) and that the chromosome 3 locus is linked with the development of paralysis. In addition, an intriguing inheritance pattern was revealed in which female backcross mice generated from B10.S female x (B10.S x SJL/J)F(1) male parents experienced significantly more EAE-related weight loss (P = 1.2 x 10(-4)) than females generated from F1 female x B10.S male parents. After controlling for this inheritance, a new locus at the centromeric end of chromosome 8 was identified that significantly influences both the development of paralysis (P = 8.2 x 10(-6)) and the incidence of CNS inflammation (P = 7.0 x 10(-5)) in EAE.

Telomere associations in interphase nuclei: possible role in maintenance of interphase chromosome topology.

The relative sizes of individual telomeres in cultured human cells under conditions of cell cycling, replicative quiescence, cell transformation and immortalization were determined using quantitative fluorescence in situ hybridization (Q-FISH) with a telomere-specific peptide nucleic acid (PNA) probe. Results obtained from analysis of telomere length profiles (TLPs), which display the distribution of relative telomere lengths for individual cells, confirmed telomere length heterogeneity at the single cell level and proportional shortening of telomere length during replicative aging of virus-transformed cells. TLPs also revealed that some telomeric ends of chromosomes are so closely juxtaposed within interphase nuclei that their fluorescent signals appear as a single spot. These telomeric associations (TAs) were far more prevalent in interphase nuclei of noncycling normal and virus-transformed cells than in their cycling counterparts. The number of interphase TAs per nucleus observed in late-passage E6/E7-transformed cells did not increase during progression to crisis, suggesting that telomere shortening does not increase the frequency of interphase TAs. Furthermore, interphase TAs were rarely observed in rapidly cycling, telomerase-positive, immortalized cells that exhibit somewhat shortened, but stabilized, telomere length through the activity of telomerase. Our overall results suggest that the number of interphase TAs is dependent more on whether or not cells are cycling than on telomere length, with TAs being most prominent in the nuclei of replicatively quiescent cells in which nonrandom (even preferred) chromosome spatial arrangements have been observed. We propose that interphase TAs may play a role in the generation and/or maintenance of nuclear architecture and chromosome positional stability in interphase nuclei, especially in cells with a prolonged G(1)/G(0) phase and possibly in terminally differentiated cells.

Gene structure and chromosomal mapping of the rat smooth muscle calponin gene.

Smooth muscle cells (SMC) express a battery of lineage-restricted genes whose encoded proteins impart the unique contractile phenotype that characterizes this muscle type. While the encoded function of many SMC-restricted genes has been extensively analyzed, less is known about their position within the genome and the regulatory factors governing their transcription. In this report, we define the gene structure, 5' promoter analysis, and chromosomal mapping of the rat smooth muscle calponin (CnnI) gene. The rat CnnI gene is comprised of seven exons spanning approximately 8 kb of genomic sequence. The intron-exon boundaries of the rat CnnI gene match precisely those in human and mouse. Primer extension and RNase protection assays indicate two major transcription start positions (tsp). Comparative sequence analysis of the 5' promoter region reveals several conserved cis regulatory elements, including a TA-rich element within 30 nt of the tsp that could be a recognition site for TATA-binding protein and two CCAAT boxes. Transient and stable transfection studies support the hypothesis that distal regulatory elements confer SMC-restricted expression of CnnI. Finally, using an F2 intercross, we have mapped the rat CnnI gene to the telomeric end of Chromosome (Chr) 8. These studies provide additional information relating to the control of CnnI gene expression and provide a platform to begin assessing the potential linkage of CnnI to spontaneous and experimental disease phenotypes in rats.

Reduction of telomeric signals in murine melanoma and human breast cancer cell lines treated with 3'-azido-2'-3'-dideoxythymidine.

The purpose of this study was to determine, using fluorescence in situ hybridization (FISH), whether treatment of cancer cells with 3'-azido-2'-3'-dideoxythymidine (AZT) has any effect on telomere length as determined by the telomeric signal intensity. To do so, we treated a metastatic murine melanoma cell line (K-1735 clone X-21) and a human breast cancer cell line (MCF-7) with three concentrations of AZT for 72 h at 37ûC. FISH preparations processed using an all-human telomeric DNA probe showed a significantly reduced, concentration-dependent telomeric signal intensity in interphase and metaphase spreads of AZT-treated cells as compared with the signal intensity in untreated controls, which showed no reduction. We conclude from these preliminary results that AZT has the potential of targeting the telomeric ends of chromosomes in cancer cells and promoting cell death and could well be tested along with other chemotherapy drugs given to cancer patients for this purpose.