Standardized protocols for differentiation of THP-1 cells to macrophages with distinct M(IFNγ+LPS), M(IL-4) and M(IL-10) phenotypes.

In vitro models of differing macrophage functions are useful since human monocyte-derived macrophages are short-lived, finite and vary from donor to donor. Published protocols using the promonocytic cell line THP-1 have tended to result in cells that closely resemble classically-activated macrophages, differentiated in IFNγ and LPS. However, no protocol, to date, has fully recapitulated polarization of THP-1 to the M(IL-4) or M(IL-10) macrophage phenotypes seen when human monocyte-derived macrophages are exposed to each cytokine. Here we present protocols that can be used to prepare M(IL-4) polarized THP-1 that transcribe CCL17, CCL26, CD200R and MRC1 and M(IL-10) cells which transcribe CD163, C1QA and SEPP1. We show that the inhibitory Fcγ Receptor IIb is preferentially expressed on the surface of M(IL-4) cells, altering the balance of activating to inhibitory Fcγ Receptors. Adoption of standardized experimental conditions for macrophage polarization will make it easier to compare downstream effector functions of different macrophage polarization states, where the impact of PMA exposure is minimized and rest periods and cytokine exposure have been optimized.

Complete Genome Sequence of a Novel Avian Paramyxovirus (APMV-13) Isolated from a Wild Bird in Kazakhstan.

A novel avian paramyxovirus was identified during annual viral surveillance of wild bird populations in Kazakhstan in 2013. The virus was isolated from a white fronted goose (Anser albifrons) in northern Kazakhstan. Here, we report the complete genome sequence of the isolate, which we suggest should constitute a novel serotype.

Both isoforms of ketohexokinase are dispensable for normal growth and development.

Dietary fructose intake has dramatically increased over recent decades and is implicated in the high rates of obesity, hypertension, and type 2 diabetes (metabolic syndrome) in Western societies. The molecular determinants of this epidemiologic correlation are incompletely defined, but high-flux fructose catabolism initiated by ketohexokinase (Khk, fructokinase) is believed to be important. The Khk gene encodes two enzyme isoforms with distinctive substrate preferences, the independent physiological roles of which are unclear. To investigate this question, and for testing the importance of Khk in metabolic syndrome, isoform-selective genetic lesions would be valuable. Two deficiency alleles of the mouse Khk gene were designed. The first, Khk(3a), uses targeted "knock-in" of a premature termination codon to induce a selective deficiency of the minor Khk-A isoform, preserving the major Khk-C isoform. The second, the Khk(Δ) allele, ablates both isoforms. Mice carrying each of these Khk-deficiency alleles were generated and validated at the DNA, RNA, and protein levels. Comparison between normal and knockout animals confirmed the specificity of the genetic lesions and allowed accurate analysis of the cellular distribution of Khk within tissues such as gut and liver. Both Khk(3a/3a) and Khk(Δ/Δ) homozygous mice were healthy and fertile and displayed minimal biochemical abnormalities under basal dietary conditions. These studies are the first demonstration that neither Khk isoform is required for normal growth and development. The new mouse models will allow direct testing of various hypotheses concerning the role of this enzyme in metabolic syndrome in humans and the value of Khk as a pharmacological target.

Inferring relative proportions of DNA variants from sequencing electropherograms.

MOTIVATION: Determination of the relative copy number of single-nucleotide sequence variants (SNVs) within a DNA sample is a frequent experimental goal. Various methods can be applied to this problem, although hybridization-based approaches tend to suffer from high-setup cost and poor adaptability, while others (such as pyrosequencing) may not be accessible to all laboratories. The potential to extract relative copy number information from standard dye-terminator electropherograms has been little explored, yet this technology is cheap and widely accessible. Since several biologically important loci have paralogous copies that interfere with genotyping, and which may also display copy number variation (CNV), there are many situations in which determination of the relative copy number of SNVs is desirable. RESULTS: We have developed a desktop application, QSVanalyzer, which allows high-throughput quantification of the proportions of DNA sequences containing SNVs. In reconstruction experiments, QSVanalyzer accurately estimated the known relative proportions of SNVs. By analyzing a large panel of genomic DNA samples, we demonstrate the ability of the software to analyze not only common biallelic SNVs, but also SNVs within a locus at which gene conversion between four genomic paralogs operates, and within another that is subject to CNV. AVAILABILITY AND IMPLEMENTATION: QSVanalyzer is freely available at http://dna.leeds.ac.uk/qsv/. It requires the Microsoft .NET framework version 2.0, which can be installed on all Microsoft operating systems from Windows 98 onwards. CONTACT: msjimc@leeds.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

BCL10 in malignant lymphomas--an evaluation using fluorescence in situ hybridization.

BCL10 is a tumour suppressor gene originally cloned from a t(1;14)(p22;q32) breakpoint in a case of mucosa-associated lymphoid tissue (MALT) lymphoma. Translocations involving this gene, though uncommon, are sometimes encountered in MALT lymphomas. This gene is thought to play an important role in the development of malignant lymphomas. Fluorescence in situ hybridization (FISH) was therefore undertaken on 22 cases of malignant lymphoma of varying histology to establish the incidence of rearrangements involving the BCL10 gene. Initially, one case with a novel t(1;2)(p22;p12) translocation involving the BCL10 gene was identified, in a marginal zone lymphoma of the MALT type, and was reported elsewhere. Seven other cases were subsequently identified with abnormalities in the 1p region, including a translocation with a breakpoint in the 1p22 region in a case of lymphoblastic lymphoma. However, none of these involved the BCL10 gene. Mutation analysis of BCL10 was then performed on 57 cases of malignant lymphoma, including 17 MALT lymphomas, by single-strand conformational polymorphism (SSCP) analysis of tumour DNA. Tissue was obtained for mutation analysis for 12 of the 22 cases analysed by FISH. Selected cases with SSCP band shifts were further studied by direct sequencing. Polymorphisms were identified in eight cases, but no mutations of pathogenic significance were identified. Further RT-PCR and mutation analysis was performed on cDNAs from 12 cases (four MALT, seven diffuse large B-cell lymphoma, one Hodgkin's disease) in which DNA analysis had already been completed. This included the MALT lymphoma with the t(1;2)(p22;p12) rearrangement. Again, no mutations were identified in the coding sequence. This study confirms that rearrangements of the BCL10 gene are uncommon in lymphoma (1/22) and may be limited tothe MALT subtype of non-Hodgkin's lymphomas. It was also found that breakpoints or rearrangements in the 1p22 region do not necessarily involve the BCL10 gene. Moreover, the absence of mutations at both the DNA (0/60) and the mRNA (0/12) level indicates that this gene is not frequently inactivated by mutation, in those tumours in which it is not involved in translocations. Our findings suggest that the BCL10 gene is unlikely to have a frequent or key role in general lymphomagenesis.

Analysis of gene expression in a human cell line stably transduced with herpesvirus saimiri.

Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus; it has significant homology to the human gammaherpesviruses Kaposi's sarcoma-associated virus and Epstein-Barr virus and the murine gammaherpesvirus murine herpesvirus 68. HVS causes a persistent asymptomatic infection in its natural host, the squirrel monkey. Both subgroups A and C possess the ability to immortalize common marmoset T lymphocytes to interleukin-2-independent proliferation. However, only subgroup C is capable of transforming human, rabbit, and rhesus monkey lymphocytes in vitro. In addition, HVS can stably transduce a variety of human cell lines where the virus persists as a nonintegrating circular episome. In this study, we have developed a system in which the HVS DNA is stably maintained as a nonintegrated circular episome in the human lung carcinoma cell line A549. Virus production can be reactivated using chemical inducing agents, including tetradecanoyl phorbol acetate and n-butyrate, suggesting that the infection in human A549 cells is latent. To analyze virus gene expression in these stably transduced cells, Northern blot analysis was performed using a series of probes produced from restriction fragments spanning the entire coding region of the HVS genome. This demonstrated that an adjacent set of genes containing open reading frames (ORFs) 71 to 73 are expressed in this stably transduced cell line. Moreover, these genes are transcribed as a polycistronic mRNA species produced from a common promoter upstream of ORF 73. This model may serve as a useful tool in the further analysis of the role of ORFs 71 to 73 in gamma-2 herpesvirus latency.

Identification of a novel microsatellite marker tightly linked to the KAI-1 gene for predicting prostate cancer progression.

BACKGROUND: We have mapped the human prostate-specific membrane antigen (PSM) gene to the chromosome 11p11.2 region at 62.5 cM, a region which also contains the prostatic cancer metastasis suppressor gene KAI-1. The genetic marker D11S1344 has been utilised for loss of heterozygosity (LOH) studies on the KAI-1 gene in a large series of prostate cancer specimens. The results were negative and it was concluded that deletions of the KAI-1 gene were not involved in the development of the metastatic phenotype in these tumours. One possible explanation for this result could be that D11S1344 is not sufficiently tightly linked to the KAI-1 gene to detect small deletions. OBJECTIVE: To attempt to identify a genetic marker more tightly linked to the KAI-1 gene than D11S1344. METHODS: Yeast artificial chromosome (YAC) clones containing the KAI-1 gene and the neighbouring marker D11S1344 were analysed by the fluorescent in situ hybridisation technique. The human genomic inserts in these novel clones were sized by pulsed field gel electrophoresis. For more accurate mapping of the KAI-1 gene, YACs containing it were screened for polymorphic markers (including D11S1344) from the 11p11.2 region. RESULTS: The novel YAC clones localised exclusively to the 11p11.2 region, with single hybridisation signals compared to the dual signals consistently obtained with nearby PSM-containing YACs. All the KAI-1 clones found had small inserts (<300 kb). The only known microsatellite which gave amplification products with these YACs was D11S986 which has been mapped at 61.3 cM on human chromosome 11. CONCLUSIONS: We have precisely localised KAI-1 at 61.3 cM on human chromosome 11. This is some 1.2 cM away from the previously utilised LOH microsatellite marker, D11S1344. We suggest that the very tightly linked microsatellite D11S986 may be a more accurate marker to assess LOH of the KAI-1 gene and thus predict progression of prostate cancer. The region of genetic duplication around the PSM gene does not extend as far distally on 11p as KAI-1.

Structural and evolutionary characterization of the human sorbitol dehydrogenase gene duplication.

We have established that two very closely homologous human sorbitol dehydrogenase sequences lie within 0.5 Mb on Chromosome 15. We have defined the relative orientation of SORD1 and SORD2 genes with respect to both the centromere and each other and established their exact chromosome location. In addition, we have identified polymorphic variants in the locus, which may be useful, in association studies to predict predisposition to clinical problems resulting from decreased conversion of cellular sorbitol to fructose. To define the evolutionary relationship of these human genes, SORD from the marmoset was also sequenced for comparison. Marmoset SORD, which appears to be a single gene in this species, shows significantly less homology with either SORD1 or SORD2 than they do with each other, suggesting that the human homologs represent a recent gene duplication event. A hypothesis is presented to explain the retention of the redundant SORD2 sequence in the human genome.

Identification and characterisation of a sequence related to human sorbitol dehydrogenase.

The polyol pathway comprises the enzymes aldose reductase and sorbitol dehydrogenase which convert glucose to fructose via sorbitol. Accumulation of sorbitol within the cell has been suggested to contribute to the progression of secondary complications of diabetes. High levels of sorbitol accumulate within the cell due to inadequate regulation of blood glucose levels. It has also been suggested that polymorphism in either the aldose reductase or sorbitol dehydrogenase genes might contribute to sorbitol accumulation. The human sorbitol dehydrogenase gene (SORD) has been described previously and a range of putative polymorphic variants were identified. Further analysis of human SORD yeast artificial chromosome clones has now shown that there is a second SORD-like sequence in man, which is extremely similar in sequence to SORD itself and which also maps to chromosome 15. Detailed sequence analysis suggests that this SORD-related gene cannot be expressed as a full-length sorbitol dehydrogenase isoenzyme. However, knowledge of the presence of this highly similar sequence in the human genome is essential to ensure that sequence variations identified during genetic analysis of SORD are not attributed to polymorphisms within that gene itself.

The herpesvirus saimiri ORF50 gene, encoding a transcriptional activator homologous to the Epstein-Barr virus R protein, is transcribed from two distinct promoters of different temporal phases.

The mRNA species encoding the herpesvirus saimiri (HVS) homolog of the Epstein-Barr virus R transcriptional activator (termed ORF50) have been identified and used to determine transcriptional start sites within the gene. The first transcript is spliced and starts from a promoter within ORF49 containing a single intron; the second is produced from a promoter within the second exon and is in the same reading frame. The spliced transcript is detected at early times during productive virus replication in OMK cells, whereas the nonspliced transcript is detected later. The spliced transcript is fivefold-more potent in activating the delayed-early ORF6 promoter; the function of the nonspliced transcript is unclear. Thus, the role of this protein in activating herpesvirus saimiri from the latent state may differ significantly from that of the Epstein-Barr virus R protein.

Yeast artificial chromosome cloning and chromosomal localization of the abundant odontogenic keratocyst protein elafin.

An imbalance in human leucocyte elastase (HLE) activity is widely recognized to play an important pathological role in a number of human diseases. An earlier report has described greater transcription of elafin, an endogenous inhibitor of HLE, in epithelia of odontogenic keratocysts of the jaw than in normal oral mucosa. The elafin gene was now localized to chromosome 20q11.2-13.1 using a combination of somatic cell-hybrid panel screening and fluorescence in situ hybridization using a biotinylated DNA probe prepared from isolated yeast artificial chromosomes. No other positive fluorescent signals were observed. This eliminates the elafin gene as a candidate gene for naevoid basal-cell carcinoma syndrome, as the gene for this syndrome localizes to chromosome 9q23.1-31. The elafin yeast artificial chromosome DNA is to be subcloned to identify polymorphic microsatellite markers that will establish whether this gene is frequently amplified in oral neoplastic tissue.

A human ubiquitin conjugating enzyme, L-UBC, maps in the Alzheimer's disease locus on chromosome 14q24.3.

We have identified a novel ubiquitin conjugating enzyme gene, L-UBC, which maps to human Chromosome (Chr) 14q24.3. This is also the location of the major early onset familial Alzheimer's disease gene (FAD3). L-UBC encodes a protein that demonstrates homology to the yeast ubiquitin conjugating enzyme, UBC-4, and human UbcH5. Their functions are to ubiquitinate specific proteins targeted for degradation. The protein also exhibits very strong homology to a rabbit protein, E2-F1, which mediates p53 degradation driven by papilloma virus E6 protein in vitro. The accumulation of specific proteins that have undergone aberrant processing in neurofibrillary tangles and amyloid plaques is the classic pathological feature in brains of Alzheimer's disease patients. Abnormal ubiquitination has previously been suggested to play a role in the etiology of Alzheimer's disease. This gene therefore represents a plausible candidate gene for FAD3.

Prostate-specific membrane antigen: evidence for the existence of a second related human gene.

Prostate-specific membrane antigen (PSM) is a glycoprotein recognised by the prostate-specific monoclonal antibody 7E11-C5, which was raised against the human prostatic carcinoma cell line LNCaP. A cDNA clone for PSM has been described. PSM is of clinical importance for a number of reasons. Radiolabelled antibody is being evaluated both as an imaging agent and as an immunotherapeutic in prostate cancer. Use of the PSM promoter has been advocated for gene therapy applications to drive prostate-specific gene expression. Although PSM is expressed in normal prostate as well as in primary and secondary prostatic carcinoma, different splice variants in malignant tissue afford the prospect of developing reverse transcription-polymerase chain reaction (RT-PCR)-based diagnostic screens for the presence of prostatic carcinoma cells in the circulation. We have undertaken characterisation of the gene for PSM in view of the protein's interesting characteristics. Unexpectedly, we have found that there are other sequences apparently related to PSM in the human genome and that PSM genomic clones map to two separate and distinct loci on human chromosome 11. Investigation of the function of putative PSM-related genes will be necessary to enable us to define fully the role of PSM itself in the development of prostatic carcinoma and in the clinical management of this malignancy.

Molecular genetic analysis of the human sorbitol dehydrogenase gene.

The polyol pathway comprises the enzymes aldose reductase and sorbitol dehydrogenase, which convert glucose to sorbitol and sorbitol to fructose, respectively, particularly in hyperglycemic states. The accumulation and toxicity of sorbitol in specific tissues has been implicated in the development of microvascular problems in some diabetic patients. Inappropriate sorbitol accumulation in some patients may be the result of polymorphic variation in the human sorbitol dehydrogenase gene, causing reduced expression levels or enzymatic activity. We now describe the structure and expression profile of the human sorbitol dehydrogenase gene and identify a range of polymorphic variants that may be useful for co-segregation studies in diabetic patients with and without severe clinical complications from their disease.

Yeast artificial chromosome cloning of the beta-catenin locus on human chromosome 3p21-22.

beta-Catenin has emerged as an important component of the adherens junctions between epithelial cells. As a result of studies of its interaction with the APC gene product, it has been implicated in the development of colorectal cancer. alpha-Catenin, beta-catenin, E-cadherin and APC appear to mediate contact inhibition in epithelia. As part of the study of the organization of the beta-catenin gene, we have isolated yeast artificial chromosomes (YACs) to characterize its intron/exon structure. YAC fluorescence in situ hybridization analysis and polymerase chain reaction analysis of somatic cell hybrid DNAs show that beta-catenin maps in the 3p21-22 region, the location of tumour-suppressor genes deleted in small-cell lung cancer (SCLC) and other disorders. beta-Catenin YACs will provide a source of microsatellite markers useful in loss of heterozygosity studies to assess the importance of beta-catenin deletions in SCLC.