Human Genomic Clones Research Articles

The evolution and population diversity of human-specific segmental duplications.

SUMMARYSegmental duplications contribute to human evolution, adaptation and genomic instability but are often poorly characterized. We investigate the evolution, genetic variation and coding potential of human-specific segmental duplications (HSDs). We identify 218 HSDs based on analysis of 322 deeply sequenced archaic and contemporary hominid genomes. We sequence 550 human and nonhuman primate genomic clones to reconstruct the evolution of the largest, most complex regions with protein-coding potential (n=80 genes/33 gene families). We show that HSDs are non-randomly organized, associate preferentially with ancestral ape duplications termed “core duplicons”, and evolved primarily in an interspersed inverted orientation. In addition to Homo sapiens-specific gene expansions (e.g., TCAF1/2), we highlight ten gene families (e.g., ARHGAP11B and SRGAP2C) where copy number never returns to the ancestral state, there is evidence of mRNA splicing, and no common gene-disruptive mutations are observed in the general population. Such duplicates are candidates for the evolution of human-specific adaptive traits.

Reconstitution of CKMT1 expression fails to rescue cells from mitochondrial membrane potential dissipation: Implications for controlling RNAi experiments

RNA interference (RNAi) is an essential method in molecular biology to reduce the expression of target genes and thereby determine their function. Since this tool is known to also have unspecific effects, control experiments are needed, chiefly among them the exclusion of off-target effects and the reconstitution of the genes' expression for the rescue of the cellular RNAi effects. We show here that the knock-down of the mitochondrial creatine kinase-1 (CKMT1) by RNA interference causes the dissipation of the mitochondrial membrane potential ΔΨm. This was accomplished with 11 different RNAi constructs designed to target 7 distinct exons as well as exon/intron junctions making unspecific off-target effects unlikely. However, all our attempts failed to rescue human cells from ΔΨm dissipation by the expression of CKMT1 alleles not targeted by RNAi. This included the transient and stable expression of the murine CKMT1 homologue, the expression of human codon usage-modified alleles, the transfection of a novel splice-isoform of CKMT1, and even the introduction of a human genomic clone for CKMT1 with codon usage changes. Our results indicate that while off-target effects of RNA interference can easily be addressed, the rescue of the knock-down phenotype is not necessarily achievable.

The Amiloride-Sensitive Endothelial Sodium Channel and Vascular Tone

See related article, pp 1053–1059 In this issue of Hypertension , Jeggle et al1 provide important new insights into the characteristics and functional significance of an amiloride-sensitive sodium channel that is expressed in vascular endothelial cells. This work extends the previous analyses by this group,2,3 and using atomic force microscopy and state-of-the art animal and cell models, provides provocative yet conclusive evidence for the central role of this channel in regulation vascular reactivity and stiffness. The Figure depicts the heteromeric structure of the amiloride-sensitive sodium channel. Although the overall topology of the channel is well described,4 there is no general agreement about the subunit composition even within epithelial cells, much less in other tissues like vascular endothelial cells that are the subject of the current report. The bulk of the subunits’ structures are expressed in the intertwined disulfide-rich extracellular domains, the role of which has not been fully explained. The subunits of the epithelial sodium channel (ENaC) were originally defined by now-classical expression cloning studies by Canessa and Rossier.5 The human genomic clones and the critical insight into the gain of function effect that characterizes Liddle syndrome6 was based on analogy to defects in the mechanosensor structures of degenerins in Caenorhabditis elegans .7 Although longitudinal flow is certainly a feature of many epithelial tubule systems, a direct connection among the putative mechanical transduction function, the extracellular domains, and regulation of apical sodium entry via ENaC is not obvious. In contrast, there are pulsatile pressures and flows in the systemic vascular system. Local regulation of vascular tone in response to changes in pressure and shear stress, an important feature of the microvascular circulation, lending credance to the idea that the extensive extracellular of the vascular endothelial …

Identification of Novel Molecular Markers for the Follow-up of Minimal Residual Disease in Hematooncological Disorders

Abstract 4905Modern molecular diagnostic techniques allow the detection of minimal residual disease (MRD) in hematological malignancies with high sensitivity, allowing patient-specific assays of MRD levels over the course of treatment. One challenge for these techniques is finding appropriate MRD marker targets. For instance, PCR-based MRD analysis requires unique sequence-specific information for the malignant clone of interest. In many acute leukemias, such detailed information is lacking, either because no abnormality is detected (e.g. in 40–50% of adult Acute myeloid leukemia-AML), or the resolution of cytogenetic methods is too low to precisely define newly-found abnormalities. We have used multicolor-FISH (mFISH) to perform karyotyping of 141 patients. Using this approach we have identified chromosomal abnormalities in about 35% of analyzed cases. The aberrations found involved the recurrent aberrations t(15;17), t(8;21), inv(16), t(9;22),−5q, −7, +8 but also an array of unique abnormalities, such as der(15)t(5;15)(q15;q24), t(6;12)(p22;q13), and der(17)t(12;17)(q14;pter), which may serve as possible targets for molecular MRD follow-up.To further characterize such targets, we seek to bridge the gap from cytogenetic resolution down to higher-resolution molecular techniques. For this, we first employ the mBAND technique, followed by multicolor FISH using DNA probes with known cytogenetic location. These probes are drawn from the Human Minimal Genomic Clone Set (Version 1.0, Source Bioscience LifeSciences) of over 25,000 bacterial artificial chromosome (BAC) clones with human DNA inserts. These are tiling clones covering almost 100% of the genome, and therefore allow us to develop FISH probes to regions containing almost any conceivable target breakpoint. Starting with breakpoint information from mFISH/mBAND, we perform consecutive multi-color/multi-BAC hybridizations around the region of interest, looking for breakpoint-spanning probes. Using this system, we are able to move from a resolution of around 2 Mbp (the limit of mBAND) to the resolution of individual BAC clones (average 150 kbp) in three hybridizations.To achieve further resolution, long-range PCR products are designed within the region defined by breakpoint-spanning BACs, with labeling and FISH-mapping of these probes. At present, we are starting to perform these hybridizations on stretched DNA (fiber-FISH or combed DNA), where one micrometer of a stretched DNA molecule represents about 2000 base pairs, allowing visualization of probe distance and spacing, and even higher-resolution breakpoint analysis. Chromosome microdissection followed by high-throughput sequencing (Roche GS Junior) is also being pursued to identify novel breakpoints by rapidly sequencing large chromosomal regions on both sides of a translocation. The final goal is to map genomic abnormalities to a resolution amenable to long-range PCR, yielding specific targets for MRD detection, and allowing clone-specific Real-Time PCR assays for sensitive and specific monitoring of MRD in hematooncological patients. Disclosures:Smolej:GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Travel Grants; Roche: Honoraria, Travel Grants; Genzyme: Honoraria, Travel Grants.

Cloning and Sequencing the First HLA Gene

This Perspectives article recounts the isolation and sequencing of the first human histocompatibility gene (HLA) in 1980-1981. At the time, general knowledge of the molecules of the immune system was already fairly extensive, and gene rearrangements in the immunoglobulin complex (discovered in 1976) had generated much excitement: HLA was quite obviously the next frontier. The author was able to use a homologous murine H-2 cDNA to identify putative human HLA genomic clones in a lambda-phage library and thus to isolate and sequence the first human histocompatibility gene. This personal account relates the steps that led to this result, describes the highly competitive international environment, and highlights the role of location, connections, and sheer luck in such an achievement. It also puts this work in perspective with a short description of the current knowledge of histocompatibility genes and, finally, presents some reflections on the meaning of "discovery."

Chromosomal evolution of the PKD1 gene family in primates.

BackgroundThe autosomal dominant polycystic kidney disease (ADPKD) is mostly caused by mutations in the PKD1 (polycystic kidney disease 1) gene located in 16p13.3. Moreover, there are six pseudogenes of PKD1 that are located proximal to the master gene in 16p13.1. In contrast, no pseudogene could be detected in the mouse genome, only a single copy gene on chromosome 17. The question arises how the human situation originated phylogenetically. To address this question we applied comparative FISH-mapping of a human PKD1-containing genomic BAC clone and a PKD1-cDNA clone to chromosomes of a variety of primate species and the dog as a non-primate outgroup species.ResultsComparative FISH with the PKD1-cDNA clone clearly shows that in all primate species studied distinct single signals map in subtelomeric chromosomal positions orthologous to the short arm of human chromosome 16 harbouring the master PKD1 gene. Only in human and African great apes, but not in orangutan, FISH with both BAC and cDNA clones reveals additional signal clusters located proximal of and clearly separated from the PKD1 master genes indicating the chromosomal position of PKD1 pseudogenes in 16p of these species, respectively. Indeed, this is in accordance with sequencing data in human, chimpanzee and orangutan. Apart from the master PKD1 gene, six pseudogenes are identified in both, human and chimpanzee, while only a single-copy gene is present in the whole-genome sequence of orangutan. The phylogenetic reconstruction of the PKD1-tree reveals that all human pseudogenes are closely related to the human PKD1 gene, and all chimpanzee pseudogenes are closely related to the chimpanzee PKD1 gene. However, our statistical analyses provide strong indication that gene conversion events may have occurred within the PKD1 family members of human and chimpanzee, respectively.ConclusionPKD1 must have undergone amplification very recently in hominid evolution. Duplicative transposition of the PKD1 gene and further amplification and evolution of the PKD1 pseudogenes may have arisen in a common ancestor of Homo, Pan and Gorilla ~8 MYA. Reticulate evolutionary processes such as gene conversion and non-allelic homologous recombination (NAHR) may have resulted in concerted evolution of PKD1 family members in human and chimpanzee and, thus, simulate an independent evolution of the PKD1 pseudogenes from their master PKD1 genes in human and chimpanzee.

Oligonucleotide Fingerprinting of Arrayed Genomic DNA Sequences Using LNA-Modified Hybridization Probes

Recently, we established a robust method for the detection of hybridization events using a DNA microarray deposited on a nanoporous membrane. Here, in a follow-up study, we demonstrate the performance of this approach on a larger set of LNA-modified oligoprobes and genomic DNA sequences. Twenty-six different LNA-modified 7-mer oligoprobes were hybridized to a set of 66 randomly selected human genomic DNA clones spotted on a nanoporous membrane slide. Subsequently, assay sensitivity analysis was performed using receiver operating characteristic (ROC) curves. Comparison of LNA-modified heptamers and DNA heptamers revealed that the LNA modification clearly improved sensitivity and specificity of hybridization experiment. Clustering analysis was applied in order to test practical performance of hybridization experiments with LNA-modified oligoprobes in recognizing similarity of genomic DNA sequences. Comparing the results with the theoretical sequence clusters, we conclude that the application of LNA-modified oligoprobes allows for reliable clustering of DNA sequences which reflects the underlying sequence homology. Our results show that LNA-modified oligoprobes can be used effectively to unravel sequence similarity of DNA sequences and thus, to characterize the content of unknown DNA libraries.

Genomic organization and expression of the human mono-ADP-ribosyltransferase ART3 gene

Here we describe an RT-PCR analysis of mono-ADP-ribosyltransferase 3 (ART3) mRNA expression in macrophages, testis, semen, tonsil, heart and skeletal muscle and the complete gene structure as obtained by sequence alignment of PCR products with a human genomic clone (GenBank accession no. AC112719). Twelve exons (ex1-12) were found to make up the coding region of the gene (one more than previously published). Two prominent classes of ART3 splice variants could be distinguished by the presence or absence of ex2 which encodes most of ART3 protein. Among the ex2-containing mRNA species, the most frequently amplified variant did not include exons 9 to 11, except in skeletal muscle, in which the major splice variant lacked ex10 only. Two different, previously not reported 5′ non-translated regions (5′ UTRs) were identified, demonstrating the presence of two alternative promoters that we termed pα and pβ. Whereas the 5′UTR originating from pα, was split up into three exons, a single exon represented the 5′ UTR of pβ transcripts. Strikingly, in heart, skeletal muscle and tonsils the upstream promoter pα was totally inactive and ART3 transcription appears to be driven solely by pβ. In all other cell types tested, transcription started mainly (if not exclusively) at pα. Thus, ART3 expression in human cells appears to be governed by a combination of differential splicing and tissue-preferential use of two alternative promoters. This specific use is evolutionary conserved as shown by analysis of the 5′ UTR of the mouse ART3 mRNA.

Expression and Packaging of a Human Endogenous Retrovirus-K Genomic DNA Clone

Human contains large number of human endogenous retroviruses (HERVs) in its genome. One of the HERV families, HERV-K, entered human genome most recently and includes many members with full-length intact proviruses. Normally, these proviruses do not express but infrequently they seem to express in cancers or autoimmune disease patients. To investigate expression mechanisms of these endogenous retroviruses, a DNA copy of HERV-K was cloned and its expression was studied. The transfection of the full-length clone into human cell lines did not produce any detectable viral capsid protein, Gag, and the transcription from its own promoter in LTR was extremely poor. The transcription was less than 10 percent compare to the exogenous retrovirus. However, when the Gag coding region was cloned under CMV promoter, Gag could be expressed efficiently and secreted as particles, probably virus like particles. The efficient expression also required a nuclear export signal. The expressed Gag could also package its own genomic RNA. These results indicate that the LTR of HERV-K is normally not active but its genes have a potential to express and possibly produce infectious particles.

Development and validation of a CGH microarray for clinical cytogenetic diagnosis

PurposeWe developed a microarray for clinical diagnosis of chromosomal disorders using large insert genomic DNA clones as targets for comparative genomic hybridization (CGH). MethodsThe array contains 362 FISH-verified clones that span genomic regions implicated in over 40 known human genomic disorders and representative subtelomeric clones for each of the 41 clinically relevant human chromosome telomeres. Three or four clones from almost all deletion or duplication genomic regions and three or more clones for each subtelomeric region were included. We tested chromosome microarray analysis (CMA) in a masked fashion by examining genomic DNA from 25 patients who were previously ascertained in a genetic clinic and studied by conventional cytogenetics. A novel software package implemented in the R statistical programming language was developed for normalization, visualization, and inference. ResultsThe CMA results were entirely consistent with previous cytogenetic and FISH findings. For clone by clone analysis, the sensitivity was estimated to be 96.7% and the specificity was 99.1%. Major advantages of this selected human genome array include the following: interrogation of clinically relevant genomic regions, the ability to test for a wide range of duplication and deletion syndromes in a single analysis, the ability to detect duplications that would likely be undetected by metaphase FISH, and ease of confirmation of suspected genomic changes by conventional FISH testing currently available in the cytogenetics laboratory. ConclusionThe array is an attractive alternative to telomere FISH and locus-specific FISH, but it does not include uniform coverage across the arms of each chromosome and is not intended to substitute for a standard karyotype. Limitations of CMA include the inability to detect both balanced chromosome changes and low levels of mosaicism.

Effect of CAT or AGG Interruptions and CpG Methylation on Nucleosome Assembly upon Trinucleotide Repeats on Spinocerebellar Ataxia, Type 1 and Fragile X Syndrome*

Nucleosome packaging regulates many aspects of DNA metabolism and is thought to mediate genetic instability and transcription of expanded trinucleotide repeats. Both instability and transcription are sensitive to repeat length, tract purity, and CpG methylation. CAT or AGG interruptions within the (CAG)n or (CGG)n tracts of spinocerebellar ataxia, type 1 or fragile X syndrome, respectively, confer increased genetic stability to the repeats. We report the formation of nucleosomes on sequences containing pure and interrupted (CAG)n and (CGG)n repeats having lengths above and below the genetic stability thresholds. Increased lengths of pure repeats led to increased and decreased propensities for nucleosome assembly on the (CAG)n and (CGG)n repeats, respectively. CpG methylation of the CGG repeat further reduced assembly. CAT interruptions in (CAG)n tracts decreased nucleosome assembly. In contrast, AGG interruptions in (CGG)n tracts did not affect assembly by hypoacetylated histones. The latter observation was unaltered by CpG methylation of the repeats. However, nucleosome assembly by hyperacetylated histones on interrupted CGG tracts was increased relative to pure tracts and this effect was abolished by CpG methylation. Thus, CAT or AGG interruptions can modulate the ability of (CAG)n and (CGG) tracts to assemble into chromatin and the effect of the AGG interruptions is dependent upon both the methylation status of the DNA and the acetylation status of the histones. Compared with the genetically unstable pure repeats, both interruptions permit a propensity of nucleosome assembly closer to that of random (genetically stable) sequences, suggesting an association of nucleosome assembly of trinucleotide repeats and genetic instability.

Exploring Williams-Beuren syndrome using myGrid.

In silico experiments necessitate the virtual organization of people, data, tools and machines. The scientific process also necessitates an awareness of the experience base, both of personal data as well as the wider context of work. The management of all these data and the co-ordination of resources to manage such virtual organizations and the data surrounding them needs significant computational infra-structure support. In this paper, we show that (my)Grid, middleware for the Semantic Grid, enables biologists to perform and manage in silico experiments, then explore and exploit the results of their experiments. We demonstrate (my)Grid in the context of a series of bioinformatics experiments focused on a 1.5 Mb region on chromosome 7 which is deleted in Williams-Beuren syndrome (WBS). Due to the highly repetitive nature of sequence flanking/in the WBS critical region (WBSCR), sequencing of the region is incomplete leaving documented gaps in the released sequence. (my)Grid was used in a series of experiments to find newly sequenced human genomic DNA clones that extended into these 'gap' regions in order to produce a complete and accurate map of the WBSCR. Once placed in this region, these DNA sequences were analysed with a battery of prediction tools in order to locate putative genes and regulatory elements possibly implicated in the disorder. Finally, any genes discovered were submitted to a range of standard bioinformatics tools for their characterization. We report how (my)Grid has been used to create workflows for these in silico experiments, run those workflows regularly and notify the biologist when new DNA and genes are discovered. The (my)Grid services collect and co-ordinate data inputs and outputs for the experiment, as well as much provenance information about the performance of experiments on WBS. The (my)Grid software is available via http://www.mygrid.org.uk

α-Pal/NRF-1 Regulates the Promoter of the Human Integrin-associated Protein/CD47 Gene

Integrin-associated protein (IAP or CD47) is expressed in a variety of tissues, including the nervous system and immune system. To understand how cells control the expression of the IAP gene, we cloned the 5'-proximal region of the human IAP gene and investigated IAP promoter activity by transient transfection. RT-PCR confirmed the expression of IAP transcripts in human neuroblastoma IMR-32 and hepatoma HepG2 cells. Deletion analysis identified a core promoter of the human IAP gene located between nucleotide positions -232 and -12 relative to the translation initiation codon in these two cell lines. Site-directed mutagenesis and gel electrophoretic mobility shift assay identified a alpha-Pal/NRF-1 binding element within the IAP core promoter. Supershift assays using the alpha-Pal/NRF-1 antiserum confirmed the binding of this transcription factor on the alpha-Pal/NRF-1 site. Overexpression of the DNA binding domain of alpha-Pal/NRF-1 in cells enhanced DNA-alpha-Pal/NRF-1 binding in vitro. Furthermore, overexpression of full-length alpha-Pal/NRF-1 significantly enhanced IAP promoter activity while overexpression of dominant-negative mutant reduced promoter activity both in the cultured human cell lines and primary mouse cortical cells. These results revealed that alpha-Pal/NRF-1 is an essential transcription factor in the regulation of human IAP gene expression.

Catalase transgenic mice: characterization and sensitivity to oxidative stress

The role of catalase in the antioxidant defense system was studied using transgenic mice [ Tg( CAT)] harboring a human genomic clone containing the entire human CAT gene. Catalase activity was 2-fold higher in the tissues of hemizygous [ Tg( CAT) +/o] mice and 3- to 4-fold higher in the tissues of homozygous [ Tg( CAT) +/+] mice compared to wild type mice. The human CAT transgene was expressed in a tissue-specific pattern that was similar to the endogenous catalase gene. The levels of other major antioxidant enzymes were not altered in the tissues of the transgenic mice. Hepatocytes and fibroblasts from the Tg( CAT) +/+ mice were more resistant to hydrogen peroxide-induced cell death but were more sensitive to paraquat and TNFα toxicity. Fibroblasts from the Tg( CAT) +/+ mice showed reduced growth rate in culture without treatment and reduced colony-forming capability after γ-irradiation compared to fibroblasts from wild type mice. In addition, the Tg( CAT) +/+ animals were more sensitive to γ-irradiation.

Cloning and characterization of the promoter region of human LPTS/PinX1 gene

The gene for LPTS/PinX1 encodes a potent telomerase inhibitor and suppresses tumor cell growth. In order to investigate the transcriptional regulation of this gene, we isolated its 5′-flanking region from the human genomic BAC clone and identified a major transcriptional initiation site. The sequence of the 5′-flanking region is GC-rich, lacks canonical TATA box, but contains potential binding sites for a variety of transcription factors. The deletion analysis indicated that the proximal 100 bp (from nt −66 to +34) is essential for minimal promoter activity and the regions of promoter from nt −1272 to −573 and nt −330 to −66 are required for maximal expression of the LPTS/PinX1 gene. Four DNase I hypersensitive sites (DHS1–4) mapping to the regions of transcription initiation and promoter in LPTS/Pinx1 gene were also revealed.

Killer cell Ig-like receptor and leukocyte Ig-like receptor transgenic mice exhibit tissue- and cell-specific transgene expression.

To generate an experimental model for exploring the function, expression pattern, and developmental regulation of human Ig-like activating and inhibitory receptors, we have generated transgenic mice using two human genomic clones: 52N12 (a 150-Kb clone encompassing the leukocyte Ig-like receptor (LILR)B1 (ILT2), LILRB4 (ILT3), and LILRA1 (LIR6) genes) and 1060P11 (a 160-Kb clone that contains ten killer cell Ig-like receptor (KIR) genes). Both the KIR and LILR families are encoded within the leukocyte receptor complex, and are involved in immune modulation. We have also produced a novel mAb to LILRA1 to facilitate expression studies. The LILR transgenes were expressed in a similar, but not identical, pattern to that observed in humans: LILRB1 was expressed in B cells, most NK cells, and a small number of T cells; LILRB4 was expressed in a B cell subset; and LILRA1 was found on a ring of cells surrounding B cell areas on spleen sections, consistent with other data showing monocyte/macrophage expression. KIR transgenic mice showed KIR2DL2 expression on a subset of NK cells and T cells, similar to the pattern seen in humans, and expression of KIR2DL4, KIR3DS1, and KIR2DL5 by splenic NK cells. These observations indicate that linked regulatory elements within the genomic clones are sufficient to allow appropriate expression of KIRs in mice, and illustrate that the presence of the natural ligands for these receptors, in the form of human MHC class I proteins, is not necessary for the expression of the KIRs observed in these mice.

DNA modification and functional delivery into human cells using Escherichia coli DH10B.

The availability of almost the complete human genome as cloned BAC libraries represents a valuable resource for functional genomic analysis, which, however, has been somewhat limited by the ability to modify and transfer this DNA into mammalian cells intact. Here we report a novel comprehensive Escherichia coli-based vector system for the modification, propagation and delivery of large human genomic BAC clones into mammalian cells. The GET recombination inducible homologous recombination system was used in the BAC host strain E.coli DH10B to precisely insert an EGFPneo cassette into the vector portion of a approximately 200 kb human BAC clone, providing a relatively simple method to directly convert available BAC clones into suitable vectors for mammalian cells. GET recombination was also used for the targeted deletion of the asd gene from the E.coli chromosome, resulting in defective cell wall synthesis and diaminopimelic acid auxotrophy. Transfer of the Yersinia pseudotuberculosis invasin gene into E.coli DH10B asd(-) rendered it competent to invade HeLa cells and deliver DNA, as judged by transient expression of green fluorescent protein and stable neomycin-resistant colonies. The efficiency of DNA transfer and survival of HeLa cells has been optimized for incubation time and multiplicity of infection of invasive E.coli with HeLa cells. This combination of E.coli-based homologous recombination and invasion technologies using BAC host strain E.coli DH10B will greatly improve the utility of the available BAC libraries from the human and other genomes for gene expression and functional genomic studies.

IL-19 induces production of IL-6 and TNF-alpha and results in cell apoptosis through TNF-alpha.

IL-10 is an immunosuppressive cytokine in the immune system. It was in clinical trial as an anti-inflammatory therapy for inflammatory bowel disease and various autoimmune diseases such as psoriasis, rheumatoid arthritis, and multiple sclerosis. IL-19 belongs to the IL-10 family, which includes IL-10, IL-19, IL-20, IL-22, melanoma differentiation-associated gene (MDA-7, IL-24), and AK155 (IL-26). Despite a partial homology in their amino acid sequences, they are dissimilar in their biologic functions. Little is known about the biologic function and gene regulation of IL-19. To understand the gene regulation of human IL-19, we identified a human IL-19 genomic clone and analyzed its promoter region. Five fusion genes containing different regions upstream of exon 1 linked to a luciferase reporter gene were expressed in the canine kidney epithelial-like Madin-Darby canine kidney cells. A fusion gene containing 394 bp showed luciferase activity 7- to 8-fold higher than the negative control of the promoterless fusion gene. We also isolated a full-length mouse cDNA clone. Mouse IL-19 shared 71% amino acid identity with human IL-19. Treatment of monocytes with mouse IL-19 induced the production of IL-6 and TNF-alpha. It also induced mouse monocyte apoptosis and the production of reactive oxygen species. Taken together, our results indicate that mouse IL-19 may play some important roles in inflammatory responses because it up-regulates IL-6 and TNF-alpha and induces apoptosis.

The SSX gene family: characterization of 9 complete genes.

Human SSX genes comprise a gene family with 6 known members. SSX1, 2 and 4 have been found to be involved in the t(X;18) translocation characteristically found in all synovial sarcomas. Four (SSX1, 2, 4 and 5) are known to be expressed in a subset of tumors and testis, and anti-SSX antibodies have been found in sera from cancer patients. SSX antigens are thus typical cancer-testis (CT) antigens. To identify additional SSX family members, we isolated and characterized human genomic clones homologous to a prototype SSX cDNA. We also searched public databases for sequences similar to SSX. This identified 3 additional SSX genes, SSX7, 8, 9, and also completed the sequence of the formerly partially defined SSX6 gene. In addition to these novel SSX genes, several SSX pseudogenes were identified. With the exception of 1 pseudogene, all SSX genomic SSX sequences map to chromosome X. Among normal tissues, SSX7 mRNA was present only in testis, whereas SSX6, 8 and 9 were not detected in any normal tissue. SSX6 and 7 were expressed in 1 of 9 melanoma cell lines tested, whereas SSX8 and 9 expression was not detected in any tumor tissue or cell lines tested. SSX1, 2, 4 and 5 mRNA expression can be induced in cell lines by 5-aza-2-deoxycytidine or Trichostatin A. These agents also induce SSX6, but not SSX3, 7, 8 or 9 in the tumor cell lines tested, indicating that mechanisms other than methylation or histone acetylation may be responsible for the repressed state of some SSX genes.

Characterization of the human prostaglandin H synthase 1 gene (PTGS1): exclusion by genetic linkage analysis as a second modifier gene in familial thrombosis.

Genetic evidence from a large Vermont kindred indicates that an unknown gene promotes thrombosis when inherited in conjunction with type I protein C deficiency. Cyclooxygenase-1 [prostaglandin H synthase 1 gene (PTGS1)] was tested as a plausible candidate for the unknown gene because of its role in primary hemostasis. The complete sequence of PTGS1 (25 638 nucleotides) was determined from a 37 kb human genomic cosmid clone to characterize intronic regions and subsequently to allow the search for mutations by direct sequencing of genomic DNA. Northern blot analysis confirms usage of a newly described distal poly-adenylation signal. Short tandem repeat (STR) sequences found in intron 2 and the 3' flanking region were developed as new genetic markers for PTGS1. The position of PTGS1 was refined on the CHLC chromosome 9 linkage map using the new markers scored in four Centre d'Etude du Polymorphisme Humain families and multipoint linkage analysis. Direct sequencing of DNA from members of the Vermont kindred led to the discovery of two new single nucleotide polymorphisms (SNPs) that give rise to non-conservative amino acid changes in the signal peptide (Arg(8) to Trp and Pro(17) to Leu) of cyclooxygenase-1. Linkage analysis of the SNP and STR markers indicated that PTGS1 is not the interacting gene associated with an increased incidence of thrombosis in the Vermont kindred.