Inhibition of HIV-1 multiplication by a modified U7 snRNA inducing Tat and Rev exon skipping

The HIV-1 regulatory proteins Tat and Rev are encoded by multiply spliced mRNAs that differ by the use of alternative 3' splice sites at the beginning of the internal exon. If these internal exons are skipped, the expression of these genes, and hence HIV-1 multiplication, should be inhibited. We have previously developed a strategy, based on antisense derivatives of U7 small nuclear RNA, that allows us to induce the skipping of an internal exon in virtually any gene. Here, we have successfully applied this approach to induce a partial skipping of the Tat, Rev (and Nef) internal exons. Three functional U7 constructs were subcloned into a lentiviral vector. Two of them strongly reduced the efficiency of lentiviral particle production compared to vectors carrying either no U7 insert or unrelated U7 cassettes. This defect could be partly or fully compensated by coexpressing Rev from an unspliced mRNA in the producing cell line. Upon stable transduction into CEM-SS or CEM T-lymphocytes, the most efficient of these constructs inhibits HIV-1 multiplication. Although the inhibition is not complete, it is more efficient in combination with another mechanism inhibiting HIV multiplication. Therefore, this new approach targeting HIV-1 regulatory genes at the level of pre-mRNA splicing, in combination with other antiviral strategies, may be a useful new tool in the fight against HIV/AIDS. Copyright (c) 2007 John Wiley & Sons, Ltd

Mutation screening of the medium-chain acyl-CoA dehydrogenase (MCAD) and the ornithine transcarbamylase (OTC) genes by multiplex PCR amplification and sequencing

BACKGROUND: Sequencing based mutation screening assays of genes encompassing large numbers of exons could be substantially optimized by multiplex PCR, which enables simultaneous amplification of many targets in one reaction. In the present study, a multiplex PCR protocol originally developed for fragment analysis was evaluated for sequencing based mutation screening of the ornithine transcarbamylase (OTC) and the medium-chain acyl-CoA dehydrogenase (MCAD) genes. METHODS: Single exon and multiplex PCR protocols were applied to generate PCR templates for subsequent DNA sequencing of all exons of the OTC and the MCAD genes. For each PCR protocol and using the same DNA samples, 66 OTC and 98 MCAD sequence reads were generated. The sequences derived from the two different PCR methods were compared at the level of individual signal-to-noise ratios of the four bases and the proportion of high-quality base-signals. RESULTS: The single exon and the multiplex PCR protocol gave qualitatively comparable results for the two genes. CONCLUSIONS: Many existing sequencing based mutation analysis protocols may be easily optimized with the proposed method, since the multiplex PCR protocol was successfully applied without any re-design of the PCR primers and other optimization steps for generating sequencing templates for the OTC and MCAD genes, respectively.

Major histocompatibility complex and other allergy-related candidate genes associated with insect bite hypersensitivity in Icelandic horses

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by bites of insects. IBH is a multifactorial disease with contribution of genetic and environmental factors. Candidate gene association analysis of IBH was performed in a group of 89 Icelandic horses all born in Iceland and imported to Europe. Horses were classified in IBH-affected and non-affected based on clinical signs and history of recurrent dermatitis, and on the results of an in vitro sulfidoleukotriene (sLT)-release assay with Culicoides nubeculosus and Simulium vittatum extract. Different genetic markers were tested for association with IBH by the Fisher's exact test. The effect of the major histocompatibility complex (MHC) gene region was studied by genotyping five microsatellites spanning the MHC region (COR112, COR113, COR114, UM011 and UMN-JH34-2), and exon 2 polymorphisms of the class II Eqca-DRA gene. Associations with Eqca-DRA and COR113 were identified (p < 0.05). In addition, a panel of 20 single nucleotide polymorphisms (SNPs) in 17 candidate allergy-related genes was tested. During the initial screen, no marker from the panel was significantly (p < 0.05) associated with IBH. Five SNPs associated with IBH at p < 0.10 were therefore used for analysis of combined genotypes. Out of them, SNPs located in the genes coding for the CD14 receptor (CD14), interleukin 23 receptor (IL23R), thymic stromal lymphopoietin (TSLP) and transforming growth factor beta 3 (TGFB3) molecules were associated with IBH as parts of complex genotypes. These results are supported by similar associations and by expression data from different horse populations and from human studies.

EGFR exon-level biomarkers of the response to bevacizumab/erlotinib in non-small cell lung cancer

Activating epidermal growth factor receptor (EGFR) mutations are recognized biomarkers for patients with metastatic non-small cell lung cancer (NSCLC) treated with EGFR tyrosine kinase inhibitors (TKIs). EGFR TKIs can also have activity against NSCLC without EGFR mutations, requiring the identification of additional relevant biomarkers. Previous studies on tumor EGFR protein levels and EGFR gene copy number revealed inconsistent results. The aim of the study was to identify novel biomarkers of the response to TKIs in NSCLC by investigating whole genome expression at the exon-level. We used exon arrays and clinical samples from a previous trial (SAKK19/05) to investigate the expression variations at the exon-level of 3 genes potentially playing a key role in modulating treatment response: EGFR, V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and vascular endothelial growth factor (VEGFA). We identified the expression of EGFR exon 18 as a new predictive marker for patients with untreated metastatic NSCLC treated with bevacizumab and erlotinib in the first line setting. The overexpression of EGFR exon 18 in tumor was significantly associated with tumor shrinkage, independently of EGFR mutation status. A similar significant association could be found in blood samples. In conclusion, exonic EGFR expression particularly in exon 18 was found to be a relevant predictive biomarker for response to bevacizumab and erlotinib. Based on these results, we propose a new model of EGFR testing in tumor and blood.

Exon involvement in the regulation of MuSVts110 RNA splicing

MuSVts110 is a conditionally defective mutant of Moloney murine sarcoma virus which undergoes a novel tmperature-dependent splice event at growth temperatures of 33$\sp\circ$C or lower. Relative to wild-type MuSV-124, MuSVts110 contains a 1487 base deletion spanning from the 3$\sp\prime$ end of the p30 gag coding region to just downstream of the first v-mos initiation codon. As a result, the gag and mos genes are fused out of frame and no v-mos protein is expressed. However, upon a shift to 33$\sp\circ$C or lower, a splice event occurs which removes 431 bases, realigns the gag and mos genes, and allows read-through translation of a P85gag-mos transforming protein. Interestingly, while the cryptic splice sites utilized in MuSVts110 are present and unaltered in MuSV-124, they are never used. Due to the 1487 base deletion, the MuSV-124 intron was reduced from 1919 to 431 bases suggesting that intron size might be involved in the activation of these cryptic splice sites in MuSVts110. Since the splicing phenotype of the MuSVts110 equivalent (TS32 DNA) which contains the identical 1487 base deletion introduced into otherwise wild-type MuSV-124 DNA, was indistinguishable from authentic MuSVts110, it was concluded that this deletion alone is responsible for activation of the cryptic splice sites used in MuSVts110. These results also confirmed that thermodependent splicing is an intrinsic property of the viral RNA and not due to some cellular defect. Furthermore, analysis of gag gene deletion and frameshift MuSVts110 mutants demonstrated that viral gag gene proteins do not play a role in regulation of MuSVts110 splicing. Instead, cis-acting viral sequences appear to mediate regulation of the splice event.^ Our initial observation that truncation of the MuSVts110 transcript, leaving only residual amounts of the flanking exon sequences, completely abolished splicing activity argued that exon sequences might participate in the regulation of the splice event.^ Analysis of exon sequence involvement has also identified cis-acting sequences important in the thermodependence of the splice event. Data suggest that regulation of the MuSVts110 splice event involves multiple interactions between specific intron and exon sequences and spliceosome components which together limit splicing activity to temperatures of 33$\sp\circ$C or lower while simultaneously restricting splicing to a maximum of 50% efficiency. (Abstract shortened with permission of author.) ^

Analysis of mutations in $\beta$-tubulin genes affecting tubulin stability and assembly

Cmd4 is a colcemid-sensitive CHO cell line that is temperature sensitive for growth and expresses an altered $\beta$-tubulin, $\beta\sb1$. One revertant of this cell line, D2, exhibits a further alteration in $\beta\sb1$ resulting in an acidic shift in its isoelectric point and a decrease in its molecular weight to 40 kD, as measured by two dimensional gel electrophoresis. This $\beta$-tubulin variant has been shown to be assembly-defective and unstable. Characterization of the mutant $\beta\sb1$ in D2 by high pressure liquid chromatography (HPLC) revealed the loss of methionine containing tryptic peptides 7,8,9, and 10. Southern analysis of the genomic DNA digested with several different restriction enzymes resulted in the appearance of new restriction fragments 250 base pairs shorter than the corresponding fragments from the wild-type $\beta\sb1$-tubulin gene. Northern analysis on mRNA from D2 revealed two new message products that also differed by 250 bases from the corresponding wild type $\beta$-tubulin transcripts. To precisely define the region of the alteration, cloning and sequencing of the mutant and wild type genomic $\beta$-tubulin genes were conducted. A size-selected EcoRI genomic library was prepared using the Stratagene lambda Zap II phage cloning system. Using subclones of CHO $\beta$-tubulin cDNA as probes, a 2.5 kb wild type clone and a 2.3 kb mutant clone were identified from this library. Each of these was shown to contain a portion of the gene extending from intron 3 through the end of the coding sequence in exon 4 and into the 3$\sp\prime$ untranslated region on the basis of alignment with the published human $\beta$-tubulin sequence. Sequencing of the mutant 2.3 kb clone revealed that the mutation is due to a 246 base pair internal deletion in exon 4 (base pair 756-1001) that encodes amino acids 253-334. This deletion results in the loss of a putative binding site for GTP which could potentially explain the phenotype of this mutant $\beta$-tubulin. Also sequence comparison of the 3$\sp\prime$ untranslated region between different species revealed the conservation of 200 base pairs with 78% homology. It is proposed that this region could play an important role in the regulation of $\beta$-tubulin gene expression. ^

Regulation of Spec genes in aboral ectoderm cells of sea urchin {\it Strongylocentrotus purpuratus}

The Spec genes of the sea urchin Stronylocentrotus purpuratus serves as an excellent model for studying cell type-specific gene expression during early embryogenesis. The Spec1/Spec2 genes encode cytosolic calcium-binding proteins related to the calmodulin/troponin C/myosin light chain superfamily. Members of the Spec gene family are activated shortly after the sixth cleavage as the lineage-specific founder cells giving rise to aboral ectoderm are established, and the accumulation of the Spec mRNAs is limited exclusively to aboral ectoderm cell lineages. In this dissertation, the transcriptional regulation of the Spec genes was studied. Sequence comparisons of the Spec gene 5$\sp\prime$ flanking regions showed that a DNA block of approximately 800 bp from the 3$\sp\prime$ end of the first exon to the 5$\sp\prime$ end of a repetitive DNA element, termed RSR, was highly conserved. In Spec2a, the conserved region was a continuous stretch of DNA, but in Spec1 and Spec2c, DNA insertions interrupt the conserved sequence block and alter the relative placement of the RSR element and other 5$\sp\prime$ flanking DNA. Thus, drastic rearrangements have occurred within the putative control regions of the Spec genes. In vivo expression experiments using the sea urchin embryo gene-transfer system showed that while the 5$\sp\prime$ flanking regions of all three Spec genes conferred proper temporal activation to the reporter CAT gene, only the Spec2a 5$\sp\prime$ flanking region could restrict lacZ gene expression to aboral ectoderm cells. However, the Spec2a conserved region alone was not sufficient to confer proper spatial expression, suggesting that negative spatial elements are also associated with the proper activation of Spec2a. A major positive regulatory region, defined as the RSR enhancer, was identified between base pairs $-$631 and $-$443 on Spec2a. The RSR enhancer was essential for maximal activity and conferred preferential aboral ectoderm expression to a lacZ reporter gene. DNaseI footprinting and band-shift analysis of the RSR enhancer revealed multiple DNA-elements. One of the elements, an A/T-rich sequence called the A/T palindrome was studied in detail. This element binds a single 45-kDa nuclear protein, the A/T palindrome binding protein (A/TBP), whose DNA-binding specificity suggests a possible relationship with the bicoid-class homeodomain proteins. Mutated A/T palindromes are incapable of binding the 45-kDa protein and lower promoter activity by 8-fold. DNA-binding activity for A/TBP is low in unfertilized eggs, increases by the 16-cell stage and continues rising in blastulae. These data suggest that A/TBP plays a major role in the activation of the Spec2a gene in aboral ectoderm cells. ^

Genomic cloning and expression of three murine UDP-galactose: beta-N-acetylglucosamine beta1,3-galactosyltransferase genes.

Based on the detection of expressed sequence tags that are similar to known galactosyltransferase sequences, we have isolated three novel UDP-galactose:beta-N-acetylglucosamine beta1, 3-galactosyltransferase (beta3GalT) genes from a mouse genomic library. The three genes, named beta3GalT-I, -II, and -III, encode type II transmembrane proteins of 326, 422, and 331 amino acids, respectively. The three proteins constitute a distinct subfamily as they do not share any sequence identity with other eucaryotic galactosyltransferases. Also, the entire protein-coding region of the three beta3GalT genes was contained in a single exon, which contrasts with the genomic organization of the beta1,4- and alpha1, 3-galactosyltransferase genes. The three beta3GalT genes were mainly expressed in brain tissue. The expression of the full-length murine genes as recombinant baculoviruses in insect cells revealed that the beta3GalT enzymes share the same acceptor specificity for beta-linked GlcNAc, although they differ in their Km for this acceptor and the donor UDP-Gal. The identification of beta3GalT genes emphasizes the structural diversity present in the galactosyltransferase gene family.

Expanding the Mutation Spectrum Affecting αIIbβ3 Integrin in Glanzmann Thrombasthenia: Screening of the ITGA2B and ITGB3 Genes in a Large International Cohort.

We report the largest international study on Glanzmann thrombasthenia (GT), an inherited bleeding disorder where defects of the ITGA2B and ITGB3 genes cause quantitative or qualitative defects of the αIIbβ3 integrin, a key mediator of platelet aggregation. Sequencing of the coding regions and splice sites of both genes in members of 76 affected families identified 78 genetic variants (55 novel) suspected to cause GT. Four large deletions or duplications were found by quantitative real-time PCR. Families with mutations in either gene were indistinguishable in terms of bleeding severity that varied even among siblings. Families were grouped into type I and the rarer type II or variant forms with residual αIIbβ3 expression. Variant forms helped identify genes encoding proteins mediating integrin activation. Splicing defects and stop codons were common for both ITGA2B and ITGB3 and essentially led to a reduced or absent αIIbβ3 expression; included was a heterozygous c.1440-13_c.1440-1del in intron 14 of ITGA2B causing exon skipping in 7 unrelated families. Molecular modeling revealed how many missense mutations induced subtle changes in αIIb and β3 domain structure across both subunits thereby interfering with integrin maturation and/or function. Our study extends knowledge of Glanzmann thrombasthenia and the pathophysiology of an integrin. This article is protected by copyright. All rights reserved.

Double-target antisense U7 snRNAs promote efficient skipping of an aberrant exon in three human beta-thalassemic mutations.

We have used three beta-thalassemic mutations, IVS2-654, -705 and -745, that create aberrant 5' splice sites (5' ss) and activate a common cryptic 3' ss further upstream in intron 2 of the human beta-globin gene to optimize a generally applicable exon-skipping strategy using antisense derivatives of U7 small nuclear RNA (snRNA). Introducing a modified U7 snRNA gene carrying an antisense sequence against the cryptic 3' ss into cultured cells expressing the mutant beta-globin genes, restored correct beta-globin mRNA splicing for all three mutations, but the efficiency was much weaker for IVS2-654 than for the other mutations. The length of antisense sequence influenced the efficiency with an optimum of approximately 24 nucleotides. Combining two antisense sequences directed against different target sites in intron 2, either on separate antisense RNAs or, even better, on a single U7 snRNA, significantly enhanced the efficiency of splicing correction. One double-target U7 RNA was expressed on stable transformation resulting in permanent and efficient suppression of the IVS2-654 mutation and production of beta-globin. These results suggest that forcing the aberrant exon into a looped secondary structure may strongly promote its exclusion from the mRNA and that this approach may be used generally to induce exon skipping.

Deletion of exon 8 from the EXT1 gene causes multiple osteochondromas (MO) in a family with three affected members.

Multiple osteochondromas (also called hereditary multiple exostoses) is an autosomal dominant disorder characterized by multiple cartilaginous tumors, which are caused by mutations in the genes for exostosin-1 (EXT1) and exostosin-2 (EXT2). The goal of this study was to elucidate the genetic alterations in a family with three affected members. Isolation of RNA from the patients' blood followed by reverse transcription and PCR amplification of selected fragments showed that the three patients lack a specific region of 90 bp from their EXT1 mRNA. This region corresponds to the sequence of exon 8 from the EXT1 gene. No splice site mutation was found around exon 8. However, long-range PCR amplification of the region from intron 7 to intron 8 indicated that the three patients contain a deletion of 4318 bp, which includes exon 8 and part of the flanking introns. There is evidence that the deletion was caused by non-homologous end joining because the breakpoints are not located within a repetitive element, but contain multiple copies of the deletion hotspot sequence TGRRKM. Exon 8 encodes part of the active site of the EXT1 enzyme, including the DXD signature of all UDP-sugar glycosyltransferases. It is conceivable that the mutant protein exerts a dominant negative effect on the activity of the EXT glycosyltransferase since it might interact with normal copies of the enzyme to form an inactive hetero-oligomeric complex. We suggest that sequencing of RNA might be superior to exome sequencing to detect short deletions of a single exon.

Molecular alterations in nucleotide excision repair genes in malignant glioma and their relevance to malignant progression and drug resistance

Recently, it has become apparent that DNA repair mechanisms are involved in the malignant progression and resistance to therapy of gliomas. Many investigators have shown that increased levels of O6-methyl guanine DNA alkyltransferase, a DNA monoalkyl adduct repair enzyme, are correlated with resistance of malignant glioma cell lines to nitrosourea-based chemotherapy. Three important DNA excision repair genes ERCC1 (excision repair cross complementation group 1), ERCC2 (excision repair cross complementation group 2), and ERCC6 (excision repair cross complementation group 6) have been studied in human tumors. Gene copy number variation of ERCC1 and ERCC2 has been observed in primary glioma tissues. A number of reports describing a relationship between ERCC1 gene alterations and resistance to anti-cancer drugs have been also described. The levels of ERCC1 gene expression, however, have not been correlated with drug resistance in gliomas. The expression of ERCC6 gene transcribes has been shown to vary with tissue types and to be highest in the brain. There have been no comprehensive studies so far, however, of ERCC6 gene expression and molecular alterations in malignant glioma. This project examined the ERCC1 expression levels and correlated them with cisplatin resistance in malignant glioma cell lines. We also examined the molecular alterations of ERCC6 gene in primary glioma tissues and cells and analyzed whether these alterations are related to tumor progression and chemotherapy resistance. Our results indicate the presence of mutations and/or deletions in exons II and V of the ERCC6 gene, and these alterations are more frequent in exon II. Furthermore, the mutations and/or deletions in exon II were shown to be associated with increased malignant grade of gliomas. The results on the Levels of ERCC1 gene transcripts showed that expression levels correlate with cisplatin resistance. The increase in ERCC1 mRNA induced by cisplatin could be down-regulated by cyclosporin A and herbimycin A. The results of this study are likely to provide useful information for clinical treatment of human gliomas. ^

Cloning, characterization and regulation of the soluble guanylyl cyclase alpha1 and beta1 genes

Cell signaling by nitric oxide (NO) through soluble guanylyl cyclase (sGC) and cGMP production regulates physiological responses such as smooth muscle relaxation, neurotransmission, and cell growth and differentiation. Although the NO receptor, sGC, has been studied extensively at the protein level, information on regulation of the sGC genes remains elusive. In order to understand the molecular mechanisms involved at the level of gene expression, cDNA and genomic fragments of the murine sGCα1 subunit gene were obtained through library screenings. Using the acquired clones, the sGCα 1 gene structure was determined following primer extension, 3 ′RACE and intron/exon boundary analyses. The basal activity of several 5′-flanking regions (putative promoter regions) for both the α1 and β1 sGC subunits were determined following their transfection into mouse N1E-115 neuroblastoma and rat RENE1Δ14 uterine epithelial cells using a luciferase reporter plasmid. Using the sGC sequences, real-time RT-PCR assays were designed to measure mRNA levels of the sGC α1 and β1 genes in rat, mouse and human. Subsequent studies found that uterine sGC mRNA and protein levels decreased rapidly in response to 17β-estradiol (estrogen) in an in vivo rat model. As early as 1 hour following treatment, mRNA levels of both sGC mRNAs decreased, and reached their lowest level of expression after 3 hours. This in vivo response was completely blocked by the pure estrogen receptor antagonist, ICI 182,780, was not seen in several other tissues examined, did not occur in response to other steroid hormones, and was due to a post-transcriptional mechanism. Additional studies ex vivo and in various cell culture models suggested that the estrogen-mediated decreased sGC mRNA expression did not require signals from other tissues, but may require cell communication or paracrine factors between different cell types within the uterus. Using chemical inhibitors and molecular targeting in other related studies, it was revealed that c-Jun-N-terminal kinase (JNK) signaling was responsible for decreased sGC mRNA expression in rat PC12 and RFL-6 cells, two models previously determined to exhibit rapid decreased sGC mRNA expression in response to different stimuli. To further investigate the post-transcriptional gene regulation, the full length sGCα1 3′-untranslated region (3′UTR) was cloned from rat uterine tissue and ligated downstream of the rabbit β-globin gene and expressed as a chimeric mRNA in the rat PC12 and RFL-6 cell models. Expression studies with the chimeric mRNA showed that the sGCα 1 3′UTR was not sufficient to mediate the post-transcriptional regulation of its mRNA by JNK or cAMP signaling in PC12 and RFL-6 cells. This study has provided numerous valuable tools for future studies involving the molecular regulation of the sGC genes. Importantly, the present results identified a novel paradigm and a previously unknown signaling pathway for sGC mRNA regulation that could potentially be exploited to treat diseases such as uterine cancers, neuronal disorders, hypertension or various inflammatory conditions. ^

Micro RNA expression alterations in patients with glioblastoma

Glioblastoma, also known as glioblastoma multiform or GBM, is the most common and most malignant primary brain tumor. The clinical history of patients with glioblastoma is short, usually less than 3 months in more than 50% of cases after diagnosis. Currently, the methods of glioblastoma treatment are chemotherapy, radiotherapy and surgery. Even with the more effective treatment options, patients with glioblastoma most likely have a median survival time of 10 to 12 months. It is necessary to seek other treatment methods, including gene-targeted treatment. The success of gene-targeted treatment depends critically on the knowledge of genes that may be the cause of, or contribute to disease. To establish a correlate between glioblastoma survival timeline and micro RNA expression alteration, a study of 91 glioblastoma patients was conducted at the University of Texas M. D. Anderson Cancer Center. These 91 glioblastoma patients were newly diagnosed from 2002 to 2007. Statistical analysis was conducted to test the association of miRNA expression alteration between long-term survival and short-term survival glioblastoma. The completion of this proposed study will provide a better understanding of the regulatory role of miRNA in glioblastoma progression.^

Identification of germ cell tumor modifier genes from the 129.MOLF-Chr19 consomic mouse strain

Naturally occurring genetic variants confer susceptibility to disease in the human population, including in testicular germ cell tumor development. Disease susceptibility loci for testicular germ cell tumors have been identified by genetic mapping in humans and mice. However, the identity of many of the susceptibility genes remains unclear. My study utilized a chromosome substitution strain, the 129.MOLF-Chr 19 (or M19 strain), to identify candidate testicular germ cell tumor susceptibility genes. Males of this strain have a high incidence of germ cell tumors in the testes. By forward genetic approaches, five susceptibility loci were fine-mapped and the genetic interactions were dissected. In addition, I identified three protein-coding genes and one micro-RNA as testicular tumor susceptibility genes by genomic screening. Using reverse genetic approaches, I verified one of the candidates, Splicing factor 1, as a modifier of testicular tumor. Deficiency of SF1 significantly reduces the incidence of testicular tumors in mice. This study highlights the advantage of the 129.MOLF-Chr 19 consomic strain in disease gene identification and validation. It also sets the stage to elucidate the molecular mechanisms of tumorigenesis in the testis. ^