APOBEC3C Tandem Domain Proteins Create Super Restriction Factors against HIV-1.

Humans encode proteins, called restriction factors, that inhibit replication of viruses such as HIV-1. The members of one family of antiviral proteins, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3; shortened here to A3), act by deaminating cytidines to uridines during the reverse transcription reaction of HIV-1. The A3 locus encodes seven genes, named A3A to A3H These genes have either one or two cytidine deaminase domains, and several of these A3s potently restrict HIV-1. A3C, which has only a single cytidine deaminase domain, however, inhibits HIV-1 only very weakly. We tested novel double domain protein combinations by genetically linking two A3C genes to make a synthetic tandem domain protein. This protein created a "super restriction factor" that had more potent antiviral activity than the native A3C protein, which correlated with increased packaging into virions. Furthermore, disabling one of the active sites of the synthetic tandem domain protein resulted in an even greater increase in the antiviral activity-recapitulating a similar evolution seen in A3F and A3G (double domain A3s that use only a single catalytically active deaminase domain). These A3C tandem domain proteins do not have an increase in mutational activity but instead inhibit formation of reverse transcription products, which correlates with their ability to form large higher-order complexes in cells. Finally, the A3C-A3C super restriction factor largely escaped antagonism by the HIV-1 viral protein Vif.IMPORTANCE As a part of the innate immune system, humans encode proteins that inhibit viruses such as HIV-1. These broadly acting antiviral proteins do not protect humans from viral infections because viruses encode proteins that antagonize the host antiviral proteins to evade the innate immune system. One such example of a host antiviral protein is APOBEC3C (A3C), which weakly inhibits HIV-1. Here, we show that we can improve the antiviral activity of A3C by duplicating the DNA sequence to create a synthetic tandem domain and, furthermore, that the proteins thus generated are relatively resistant to the viral antagonist Vif. Together, these data give insights about how nature has evolved a defense against viral pathogens such as HIV.

Association of Platelet Integrin αIIbß3 Polymorphisms with Atherosclerotic Coronary Heart Disease in Sudanese Patients.

BACKGROUND AND OBJECTIVE: Glycoprotein IIIa and GPIIb constitutes are the fibrinogen receptor (integrin αIIbß3). This glycoprotein has a fundamental role in atherothrombosis. This study aimed to detect the association of αIIbß3 polymorphisms with atherosclerotic coronary heart disease in Sudanese patients and the association between the risk factors and platelet integrin αIIbß3 polymorphisms. MATERIALS AND METHODS: This is a case-control hospital-based study contain 50 atherosclerotic patients (>18 years) with coronary heart disease that admitted to Khartoum hospital and were compared to apparently 50 healthy Sudanese subjects at the same ages. About 5 mL of venous blood sample was collected from each patient and control. The laboratory analyses were done for HbA1c, lipid profile and for DNA genotyping. RESULTS: LDL, HDL, HbA1c and body mass index have shown highly significant influence on patients. No significant differences were observed for triglycerides and total cholesterol levels. The risks of coronary heart disease were higher with A/B genotype in HPA3, but no association detected with coronary heart disease patients in HPA1 polymorphism. CONCLUSION: In conclusion, HPA3 polymorphism was associated with atherosclerotic in Sudanese patients, while HPA1 polymorphism has not.

The Use of Methylation-Sensitive Multiplex Ligation-Dependent Probe Amplification for Quantification of Imprinted Methylation.

Imprinting disorders are a group of congenital diseases that can result from multiple mechanisms affecting imprinted gene dosage including cytogenetic aberration and epigenetic anomalies. Quantification of CpG methylation and correct copy-number calling is required for molecular diagnosis. Methylation-sensitive multiplex ligation-dependent probe amplification (MS-MLPA) is a multiplex method that accurately measures both parameters in a single assay. This technique relies upon the ligation of MLPA probe oligonucleotides and digestion of the genomic DNA-probe hybrid complexes with the Hha1 methylation-sensitive restriction endonuclease prior to fluorescent PCR amplification with a single primer pair. Since each targeted probe contains stuffer sequence of varying length, each interrogated position is visualized as an amplicon of different size upon capillary electrophoresis.

Damaging the Integrated HIV Proviral DNA with TALENs.

HIV-1 integrates its proviral DNA genome into the host genome, presenting barriers for virus eradication. Several new gene-editing technologies have emerged that could potentially be used to damage integrated proviral DNA. In this study, we use transcription activator-like effector nucleases (TALENs) to target a highly conserved sequence in the transactivation response element (TAR) of the HIV-1 proviral DNA. We demonstrated that TALENs cleave a DNA template with the HIV-1 proviral target site in vitro. A GFP reporter, under control of HIV-1 TAR, was efficiently inactivated by mutations introduced by transfection of TALEN plasmids. When infected cells containing the full-length integrated HIV-1 proviral DNA were transfected with TALENs, the TAR region accumulated indels. When one of these mutants was tested, the mutated HIV-1 proviral DNA was incapable of producing detectable Gag expression. TALEN variants engineered for degenerate recognition of select nucleotide positions also cleaved proviral DNA in vitro and the full-length integrated proviral DNA genome in living cells. These results suggest a possible design strategy for the therapeutic considerations of incomplete target sequence conservation and acquired resistance mutations. We have established a new strategy for damaging integrated HIV proviral DNA that may have future potential for HIV-1 proviral DNA eradication.

Formation of carcinogenic chromosomal rearrangements in human thyroid cells after induction of double-strand DNA breaks by restriction endonucleases.

Ionizing radiation (IR) exposure increases the risk of thyroid cancer and other cancer types. Chromosomal rearrangements, such as RET/PTC, are characteristic features of radiation-associated thyroid cancer and can be induced by radiation in vitro. IR causes double-strand breaks (DSBs), suggesting that such damage leads to RET/PTC, but the rearrangement mechanism has not been established. To study the mechanism, we explored the possibility of inducing RET/PTC by electroporation of restriction endonucleases (REs) into HTori-3 human thyroid cells. We used five REs, which induced DSB in a dose-dependent manner similar to that seen with IR. Although all but one RE caused DSB in one or more of the three genes involved in RET/PTC, rearrangement was detected only in cells electroporated with either PvuII (25 and 100  U) or StuI (100 and 250  U). The predominant rearrangement type was RET/PTC3, which is characteristic of human thyroid cancer arising early after Chernobyl-related radioactive iodine exposure. Both enzymes that produced RET/PTC had restriction sites only in one of the two fusion partner genes. Moreover, the two enzymes that produced RET/PTC had restriction sites present in clusters, which was not the case for RE that failed to induce RET/PTC. In summary, we establish a model of DSB induction by RE and report for the first time the formation of carcinogenic chromosomal rearrangements, predominantly RET/PTC3, as a result of DSB produced by RE. Our data also raise a possibility that RET/PTC rearrangement can be initiated by a complex DSB that is induced in one of the fusion partner genes.

Isolation and characterization of a new bacteriophage MMP17 from Meiothermus.

Thermophiles and their viruses are extraordinarily important because of their roles in processes of evolution, biogeochemistry and ecology. Species of the genus Meiothermus share close relationship with genus Thermus, but no Meiothermus bacteriophage has been reported yet. In this work, a new thermophilic bacteriophage named MMP17 (Meiothermus Myoviridae phage 17) was isolated from a Meiothermus strain and was further characterized. MMP17 was a typical myovirus with an icosahedral head (42 nm in diameter) and a tail (120 nm in length and 17 nm in width). Its DNA was about 33.5-39.5 kb in size. MMP17 was very stable at 55-60°C and pH 6-7. According to the one-step growth curve, the latent period and the burst period were 60 and 30 min, respectively. An average of 15 phage particles was released from each infected cell. Four major bands were detected in purified virion preparation by SDS-PAGE. As MMP17 was a thermophilic bacteriophage with lower production temperature, its characterization and the relationship between MMP17 and Thermus bacteriophages deserved for further study.

Methylation of histone H3 lysine 36 enhances DNA repair by nonhomologous end-joining.

Given its significant role in the maintenance of genomic stability, histone methylation has been postulated to regulate DNA repair. Histone methylation mediates localization of 53BP1 to a DNA double-strand break (DSB) during homologous recombination repair, but a role in DSB repair by nonhomologous end-joining (NHEJ) has not been defined. By screening for histone methylation after DSB induction by ionizing radiation we found that generation of dimethyl histone H3 lysine 36 (H3K36me2) was the major event. Using a novel human cell system that rapidly generates a single defined DSB in the vast majority of cells, we found that the DNA repair protein Metnase (also SETMAR), which has a SET histone methylase domain, localized to an induced DSB and directly mediated the formation of H3K36me2 near the induced DSB. This dimethylation of H3K36 improved the association of early DNA repair components, including NBS1 and Ku70, with the induced DSB, and enhanced DSB repair. In addition, expression of JHDM1a (an H3K36me2 demethylase) or histone H3 in which K36 was mutated to A36 or R36 to prevent H3K36me2 formation decreased the association of early NHEJ repair components with an induced DSB and decreased DSB repair. Thus, these experiments define a histone methylation event that enhances DNA DSB repair by NHEJ.

Protoplast transformation of filamentous fungi.

The protoplast method for the transformation of filamentous fungi is described in detail, as is the Restriction Enzyme-Mediated Integration (REMI) procedure for introducing tagged mutations into the fungal genome. A split marker method for generating PCR fragments for targeted integration and deletion of genes of interest is also detailed.

Characterization of erythropoietin receptor and erythropoietin expression and function in human ovarian cancer cells.

The identification of erythropoietin receptors (EpoR) on cancer cells has caused concern, since it implies the possibility that treatment of cancer patients with erythropoietin (Epo) and related agents with demonstrable antiapoptotic activity could enhance cancer growth and progression. However, the function and even the validity of the identification of these receptors have been called into question. We now report the characterization of EpoR and Epo expression by 4 human ovarian cancer cell lines: A2780, CaOV, SKOV and OVCAR-3. Using semiquantitative RT-PCR, restriction digestion of the PCR products and DNA sequence analysis, we determined that each of the lines expresses the EpoR and Epo at the mRNA level. A2780 cells were the highest expressers of both genes. We demonstrated EpoR protein both by western blotting and by immunofluorescence and biologically active Epo protein by quantitative in vitro bioassay. The EpoR on A2780 cells was shown to be functional, since Epo stimulation resulted in phosphorylation of Erk1/2, an important EpoR mitogenic signaling intermediate. None of the cell lines exhibited a growth response in culture to exogenous Epo. However, addition of a neutralizing anti-Epo antibody to A2780 cells resulted in partial growth inhibition that was reversed by the addition of excess Epo, providing evidence for an autocrine/paracrine mechanism of growth enhancement in these cells.

Murine gammaherpesvirus 68 open reading frame 24 is required for late gene expression after DNA replication.

Open reading frame 24 (ORF24) of murine gammaherpesvirus 68 (MHV-68) is conserved among beta- and gammaherpesviruses; however, its function in viral replication has not been defined. Using MHV-68 as a model, we have identified ORF24 as being essential for viral replication. An ORF24-null virus was generated and shown to be defective in late gene expression. Expression of early genes, as well as viral genome replication, was not affected. Furthermore, the defect in late gene expression was likely due to a deficiency in transcription. Thus, we have identified an MHV-68 protein, ORF24, that is essential for the expression of viral late proteins yet dispensable for viral DNA replication.

Homozygosity for the double D409H+H255Q allele in type II Gaucher disease.

Homozygosity for D409H has been associated with a unique type III subtype of the disease with a phenotype dominated by severe cardiovascular involvement, whereas neurological findings, if present, are restricted to oculomotor apraxia and features such as visceromegaly are either minimal or absent. Using PCR amplification followed by restriction enzyme analysis, 3 patients (1 Greek, 2 Albanians) were IDentified with the D409H/D409H genotype. All shared a very severe early-onset neurological phenotype that classified them as type II. Amplification and sequencing of the full coding region of the GBA gene revealed that all three patients were homozygous not only for D409H but also for H255Q. Both mutations were present on the same allele, as shown by analysis of the parental DNA. The double D409H+H255Q allele was found in heterozygosity in Greek, Bulgarian and Argentinian patients but was not IDentified in any Spanish patients carrying the D409H mutation.

Analysis of DNA methylation profiles in preimplantation embryos using bisulfite mutagenesis.

For developmental competence of mammalian embryos, dynamic epigenetic changes to both the maternally and paternally derived contributions to the genome of the zygote should be brought about in the early cleavage stages. DNA methylation is a typical epigenetic mark modified during pre-implantation development. Here, we describe how to analyze DNA methylation profiles in early-stage embryos.

DNA sequence analysis of a small cryptic plasmid from Lactococcus lactis subsp. lactis M14.

A small plasmid designated pAR141 was isolated from Lactococcus lactis subsp. lactis M14 and its complete 1,594 base pair nucleotide sequence was determined. Analysis of the sequence indicated that this plasmid does not carry any industrially important determinants besides the elements involved in plasmid replication and control. The transcriptional repressor CopG and replication initiation protein RepB appeared as a single operon. A small countertranscribed RNA (ctRNA) coding region was found between the copG and repB genes. The double strand origin (dso) and single strand origin (sso) of rolling circle replicating (RCR) plasmids were also identified in pAR141, suggesting that this plasmid replicates by rolling circle (RC) mode. This observation was supported by S1 nuclease and Southern hybridization analyses.

Molecular classification of IncP-9 naphthalene degradation plasmids.

A large collection of naphthalene-degrading fluorescent Pseudomonas strains isolated from sites contaminated with coal tar and crude oil was screened for the presence of IncP-9 plasmids. Seventeen strains were found to carry naphthalene catabolic plasmids ranging in size from 83 to 120 kb and were selected for further study. Results of molecular genotyping revealed that 15 strains were closely related to P. putida, one to P. fluorescens, and one to P. aeruginosa. All catabolic plasmids found in these strains, with the exception of pBS216, pSN11, and p8909N-1, turned out to belong to IncP-9 beta-subgroup. Plasmids pBS216, pSN11, and p8909N-1 were identified as members of IncP-9 delta-subgroup. One plasmid, pBS2, contains fused replicons of IncP-9beta and IncP-7 groups. RFLP analyses of the naphthalene catabolic plasmids revealed that organisation of the replicon correlates well with the overall plasmid structure. Comparative PCR studies with conserved oligonucleotide primers indicated that genes for key enzymes of naphthalene catabolism are highly conserved among all studied plasmids. Three bacterial strains, P. putida BS202, P. putida BS3701, and P. putida BS3790, were found to have two different salicylate hydroxylase genes one of which has no similarity to the "classic" enzyme encoded by nahG gene. Discovery of a large group of plasmid with unique nahR suggested that the regulatory loop may also represent a variable part of the pathway for catabolism of naphthalene in fluorescent Pseudomonas spp.

Interleukin-21 receptor gene induction in human T cells is mediated by T-cell receptor-induced Sp1 activity.

Interleukin-21 (IL-21) plays important roles in regulating the immune response. IL-21 receptor (IL-21R) mRNA is expressed at a low level in human resting T cells but is rapidly induced by mitogenic stimulation. We now investigate the basis for IL21R gene regulation in T cells. We found that the -80 to -20 region critically regulates IL-21R promoter activity and corresponds to a major DNase I-hypersensitive site. Electrophoretic mobility shift assays, DNA affinity chromatography followed by mass spectrometry, and chromatin immunoprecipitation assays revealed that Sp1 binds to this region in vitro and in vivo. Moreover, mutation of the Sp1 motif markedly reduced IL-21R promoter activity, and Sp1 small interfering RNAs effectively diminished IL-21R expression in activated T cells. Interestingly, upon T-cell receptor (TCR) stimulation, T cells increased IL-21R expression and Sp1 protein levels while decreasing Sp1 phosphorylation. Moreover, phosphatase inhibitors that increased phosphorylation of Sp1 diminished IL-21R transcription. These data indicate that TCR-induced IL-21R expression is driven by TCR-mediated augmentation of Sp1 protein levels and may partly depend on the dephosphorylation of Sp1.

Repair-independent chromatin assembly onto active ribosomal genes in yeast after UV irradiation.

Chromatin rearrangements occur during repair of cyclobutane pyrimidine dimers (CPDs) by nucleotide excision repair (NER). Thereafter, the original structure must be restored to retain normal genomic functions. How NER proceeds through nonnucleosomal chromatin and how open chromatin is reestablished after repair are unknown. We analyzed NER in ribosomal genes (rDNA), which are present in multiple copies but only a fraction are actively transcribed and nonnucleosomal. We show that removal of CPDs is fast in the active rDNA and that chromatin reorganization occurs during NER. Furthermore, chromatin assembles on nonnucleosomal rDNA during the early events of NER but in the absence of DNA repair. The resumption of transcription after removal of CPDs correlates with the reappearance of nonnucleosomal chromatin. To date, only the passage of replication machinery was thought to package ribosomal genes in nucleosomes. In this report, we show that early events after formation of UV photoproducts in DNA also promote chromatin assembly.

The DNA demethylating 5-azaC induces endoreduplication in cultured Chinese hamster cells.

We have investigated the possible influence of 5-azacytidine (5-azaC) substitution for cytidine into DNA on topoisomerase II (topo II) function in chromosome segregation. The endpoint chosen has been the induction of endoreduplicated cells at mitosis showing diplochromosomes. Experiments were performed in the presence and absence of the cytidine analogue to assess the degree of 5-azaC-induced DNA hypomethylation, using differential cutting by restriction endonucleases Hpa II and Msp I. Using the pulsed-field gel electrophoresis (PFGE) technique, we have also observed a protective effect provided by 5-azaC treatment against DNA breakage induced by the topo II poison m-AMSA. Concentrations of 5-azaC shown as able to induce extensive DNA hypomethylation and capable to protect DNA from double-strand breaks induced by m-AMSA were used for our cytogenetic experiments to analyze chromosome segregation. Our results seem to indicate that the presence of 5-azaC in DNA induces a dose-dependent increase in the yield of endoreduplicated cells that parallels the levels of hypomethylation observed.

Specific ion effects on the growth rates of Staphylococcus aureus and Pseudomonas aeruginosa.

Motivated by recent advances in the physical and chemical basis of the Hofmeister effect, we measured the rate cell growth of S. aureus--a halophilic pathogenic bacterium--and of P. aeruginosa, an opportunistic pathogen, in the presence of different aqueous salt solutions at different concentrations (0.2, 0.6 and 0.9 M). Microorganism growth rates depend strongly on the kind of anion in the growth medium. In the case of S. aureus, chloride provides a favorable growth medium, while both kosmotropes (water structure makers) and chaotropes (water structure breakers) reduce the microorganism growth. In the case of P. aeruginosa, all ions affect adversely the bacterial survival. In both cases, the trends parallel the specific ion, or Hofmeister, sequences observed in a wide range of physico-chemical systems. The correspondence with specific ion effect obtained in other studies, on the activities of a DNA restriction enzyme, of horseradish peroxidase, and of Lipase A (Aspergillus niger) is particularly striking. This work provides compelling evidence for Hofmeister effects, physical chemistry in action, in these organisms.

Description of a PCR-based technique for DNA splicing and mutagenesis by producing 5' overhangs with run through stop DNA synthesis utilizing Ara-C.

BACKGROUND: Splicing of DNA molecules is an important task in molecular biology that facilitates cloning, mutagenesis and creation of chimeric genes. Mutagenesis and DNA splicing techniques exist, some requiring restriction enzymes, and others utilize staggered reannealing approaches. RESULTS: A method for DNA splicing and mutagenesis without restriction enzymes is described. The method is based on mild template-dependent polymerization arrest with two molecules of cytosine arabinose (Ara-C) incorporated into PCR primers. Two rounds of PCR are employed: the first PCR produces 5' overhangs that are utilized for DNA splicing. The second PCR is based on polymerization running through the Ara-C molecules to produce the desired final product. To illustrate application of the run through stop mutagenesis and DNA splicing technique, we have carried out splicing of two segments of the human cofilin 1 gene and introduced a mutational deletion into the product. CONCLUSION: We have demonstrated the utility of a new PCR-based method for carrying out DNA splicing and mutagenesis by incorporating Ara-C into the PCR primers.

Promoter hypermethylation is the predominant mechanism in hMLH1 and hMSH2 deregulation and is a poor prognostic factor in nonsmoking lung cancer.

PURPOSE AND EXPERIMENTAL DESIGN: The etiologic association and prognostic significance of mismatch repair gene/protein alterations have never been examined in nonsmoking lung cancer. Therefore, we investigated protein expression and promoter hypermethylation of hMLH1 and hMSH2 genes in the tumor specimens from 105 nonsmoking female non-small cell lung cancer (NSCLC) patients. Immunohistochemistry and restriction enzyme-based multiplex PCR were used to examine the protein expression and promoter hypermethylation, respectively. The occurrence of gene/protein alteration for each gene was compared with the patients' clinicopathologic variables as well as the overall survival and cancer-specific survival rates. RESULTS: Protein expression alteration and promoter hypermethylation were observed in 66% to 67% and 30% to 34% of tumor specimens for hMLH1 and hMSH2 genes, respectively. Loss of hMLH1 and hMSH2 protein expression was significantly associated with their promoter hypermethylation (P < 0.0001 and P = 0.049). The overall survival and cancer-specific survival rates were significantly lower in patients with promoter hypermethylation of hMSH2 gene than in those without hypermethylation (P = 0.038 and P = 0.004). The poor prognosis was still especially significant in adenocarcinoma (P = 0.035 and P = 0.061) and early-stage NSCLC patients (P = 0.067 and P = 0.041). CONCLUSION: Our data suggest that hMLH1 is the major altered mismatch repair gene involved in nonsmoking NSCLC tumorigenesis and that promoter methylation is the predominant mechanism in hMLH1 and hMSH2 deregulation. In addition, promoter methylation of the hMSH2 gene may be a potential prognostic factor in nonsmoking female lung cancer.