Detecting AIDS restriction genes: from candidate genes to genome-wide association discovery.

The screening of common genetic polymorphisms among candidate genes for AIDS pathology in HIV exposed cohort populations has led to the description of 20 AIDS restriction genes (ARGs), variants that affect susceptibility to HIV infection or to AIDS progression. The combination of high-throughput genotyping platforms and the recent HapMap annotation of some 3 million human SNP variants has been developed for and applied to gene discovery in complex and multi-factorial diseases. Here, we explore novel computational approaches to ARG discovery which consider interacting analytical models, various genetic influences, and SNP-haplotype/LD structure in AIDS cohort populations to determine if these ARGs could have been discovered using an unbiased genome-wide association approach. The procedures were evaluated by tracking the performance of haplotypes and SNPs within ARG regions to detect genetic association in the same AIDS cohort populations in which the ARGs were originally discovered. The methodology captures the signals of multiple non-independent AIDS-genetic association tests of different disease stages and uses association signal strength (odds ratio or relative hazard), statistical significance (p-values), gene influence, internal replication, and haplotype structure together as a multi-facetted approach to identifying important genetic associations within a deluge of genotyping/test data. The complementary approaches perform rather well and predict the detection of a variety of undiscovered ARGs that affect different stages of HIV/AIDS pathogenesis using genome-wide association analyses.

Markers for mapping by admixture linkage disequilibrium in African American and Hispanic populations.

Population linkage disequilibrium occurs as a consequence of mutation, selection, genetic drift, and population substructure produced by admixture of genetically distinct ethnic populations. African American and Hispanic ethnic groups have a history of significant gene flow among parent groups, which can be of value in affecting genome scans for disease-gene discovery in the case-control and transmission/disequilibrium test designs. Disease-gene discovery using mapping by admixture linkage disequilibrium (MALD) requires a map of polymorphic markers that differentiate between the founding populations, along with differences in disease-gene allele frequencies. We describe markers appropriate for MALD mapping by assessing allele frequencies of 744 short tandem repeats (STRs) in African Americans, Hispanics, European Americans, and Asians, by choosing STR markers that have large differences in composite delta, log-likelihood ratios, and/or I*(2) for MALD. Additional markers can be added to this MALD map by utilization of the rapidly growing single-nucleotide-polymorphism databases and the literature, to achieve a 3-10-cM scanning scale. The map will be useful for studies of diseases, including prostate and breast cancer, diabetes, hypertension, and end-stage renal disease, that have large differences in incidence between the founding populations of either Hispanics or African Americans.

Significant admixture linkage disequilibrium across 30 cM around the FY locus in African Americans.

Scientists, to understand the importance of allelic polymorphisms on phenotypes that are quantitative and environmentally interacting, are now turning to population-association screens, especially in instances in which pedigree analysis is difficult. Because association screens require linkage disequilibrium between markers and disease loci, maximizing the degree of linkage disequilibrium increases the chances of discovering functional gene-marker associations. One theoretically valid approach-mapping by admixture linkage disequilibrium (MALD), using recently admixed African Americans-is empirically evaluated here by measurement of marker associations with 15 short tandem repeats (STRs) and an insertion/deletion polymorphism of the AT3 locus in a 70-cM segment at 1q22-23, around the FY (Duffy) locus. The FY polymorphism (-46T-->C) disrupts the GATA promoter motif, specifically blocking FY erythroid expression and has a nearly fixed allele-frequency difference between European Americans and native Africans that is likely a consequence of a selective advantage of FY-/- in malaria infections. Analysis of linkage disequilibrium around the FY gene has indicated that there is strong and consistent linkage disequilibrium between FY and three flanking loci (D1S303, SPTA1, and D1S484) spanning 8 cM. We observed significant linkage-disequilibrium signals over a 30-cM region from -4.4 to 16.3 cM (from D1S2777 to D1S196) for STRs and at 26.4 cM (AT3), which provided quantitative estimates of centimorgan limits, by MALD assessment in African American population-association analyses, of 5-10 cM.

A human genome map of comparative anchor tagged sequences.

Effective comparative mapping inference utilizing developing gene maps of animal species requires the inclusion of anchored reference loci that are homologous to genes mapped in the more "gene-dense" mouse and human maps. Nominated anchor loci, termed comparative anchor tagged sequences (CATS), have been ordered in the mouse linkage map, but due to the dearth of common polymorphisms among human coding genes have not been well represented in human linkage maps. We present here an ordered framework map of 314 comparative anchor markers in humans based on mapping analysis in the Genebridge 4 panel of radiation hybrid cell lines, plus empirically optimized CATS PCR primers which detect these markers. The ordering of these homologous gene markers in human and mouse maps provides a framework for comparative gene mapping of representative mammalian species.

Effect of oncogene expression on telomerase activation and telomere length in human endothelial, fibroblast and prostate epithelial cells.

Although strong evidence is mounting that telomerase reactivation and the thereof resulting stabilization of telomeres is a major mechanism for human cells to overcome replicative senescence, a causal relationship linking telomerase activation conclusively to tumorigenesis remains to be established. Thus, the possibility exists that telomerase activation is passively co-selected as tumors develop. To elucidate the function of telomerase during tumorigenesis, we followed telomerase reactivation during immortalization of human primary cell types with in vitro transforming agents and determined the tumorigenic potential of these cells at various stages of transformation. The effects of SV40, v-Ki-ras, HPV-18 and HPV-16 E6/E7 oncoproteins on telomerase expression was examined in primary and immortalized human prostate epithelial (HPE), human prostate fibroblast (HPF), and umbilical vein endothelial cells (HUVEC). All of five SV40-transformed HPE and HPF lines were telomerase positive and had shorter telomeres than primary cells. The two HPV-18 immortalized HPE cell lines also expressed telomerase activity. In contrast, E6 or E7 alone could not produce immortalized HUVEC and did not reactivate telomerase. Life-span, however, was extended. The E6/E7 immortalized HUVEC had telomerase activity and short but stable telomeres. HPE, HPF or HUVEC cells which had been transformed by one oncoprotein alone were not tumorigenic although they had overcome cellular senescence and re-activated telomerase. However, if these cells were transformed by a second agent, either infection with v-Ki-ras or X-ray treatment, they were able to form tumors in nude mice. This suggests that tumorigenesis is a multistep process and that telomerase activation alone is not sufficient for malignant transformation in human cells.

Binding of c-Raf1 kinase to a conserved acidic sequence within the carboxyl-terminal region of the HIV-1 Nef protein.

Nef is a membrane-associated cytoplasmic phosphoprotein that is well conserved among the different human (HIV-1 and HIV-2) and simian immunodeficiency viruses and has important roles in down-regulating the CD4 receptor and modulating T-cell signaling pathways. The ability to modulate T-cell signaling pathways suggests that Nef may physically interact with T-cell signaling proteins. In order to identify Nef binding proteins and map their site(s) of interaction, we targeted a highly conserved acidic sequence at the carboxyl-terminal region of Nef sharing striking similarity with an acidic sequence at the c-Raf1-binding site within the Ras effector region. Here, we used deletion and site-specific mutagenesis to generate mutant Nef proteins fused to bacterial glutathione S-transferase in in vitro precipitation assays and immunoblot analysis to map the specific interaction between the HIV-1LAI Nef and c-Raf1 to a conserved acidic sequence motif containing the core sequence Asp-Asp-X-X-X-Glu (position 174-179). Significantly, we demonstrate that substitution of the nonpolar glycine residue for either or both of the conserved negatively charged aspartic acid residues at positions 174 and 175 in the full-length recombinant Nef protein background completely abrogated binding of c-Raf1 in vitro. In addition, lysates from a permanent CEM T-cell line constitutively expressing the native HIV-1 Nef protein was used to coimmunoprecipitate a stable Nef-c-Raf1 complex, suggesting that molecular interactions between Nef and c-Raf1, an important downstream transducer of cell signaling through the c-Raf1-MAP kinase pathway, occur in vivo. This interaction may account for the Nef-induced perturbations of T-cell signaling and activation pathways in vitro and in vivo.

A human vascular endothelial cell model to study angiogenesis and tumorigenesis.

Endothelial cell biology has recently been the subject of considerable interest in thrombosis and cancer research. However, the successful establishment of immortalized human endothelial cells which retain differentiated cell characteristics has been rare. We have successfully established immortalized human umbilical vein endothelial cells (HUVECs) by human papilloma virus (HPV)-16 E6-E7. HPV-16 E6, E7 and E6-E7 were successfully introduced into HUVEC cells. Both E6 and E7 cultures had an extended lifespan but eventually underwent senescence. E6-E7 cultures 4-5-2G, however, acquired an indefinite lifespan in culture but did not undergo malignant conversion. Telomerase activity was not detected in either E6 or E7 cultures; however, telomerase was detected in E6-E7 4-5-2G cells. The cells exhibited a 'cobblestone' morphology and developed a capillary-like tube structure upon reaching confluence. The 4-5-2G line expressed Factor VIII related antigen and took up DiI-Ac-LDL as markers of endothelial origin. The line expressed integrin subunits (alpha(v)beta3, alph(v)beta5, beta1, alpha2, alpha3, beta4 and alpha6) consistent with an endothelial origin. The higher passage of 4-5-2G line showed a similar intensity of integrin immunostaining to that of primary HUVECS. Subsequent infection of these immortal cells with the Kirsten murine sarcoma virus which contains an activated K-ras oncogene induced morphological transformation that led to the acquisition of invasion capability and neoplastic properties. Telomerase was also detected in the tumorigenic v-Ki-ras transformed cell line. These cell lines should be useful for studies of the molecular mechanisms underlying normal and neoplastic endothelial cell proliferation and migration, and might also provide an in vitro model for development of pharmacologic and gene therapy for cardiovascular thrombosis and cancer.

ETS-1 induces increased expression of erythroid markers in the pluripotent erythroleukemic cell lines K562 and HEL.

Members of the ETS gene family are known to be expressed in hematopoietic tissues and cell lines, and there is increasing evidence that ETS proteins may play a role in normal hematopoietic cell development. We demonstrate that ETS-1 can contribute to the development of an erythroid phenotype in vitro. The pluripotent erythroleukemic K562 and HEL cell lines express messages for a number of ETS genes, but only c-ETS-1 levels are elevated in response to treatment with hemin or cytosine arabinofuranoside (Ara-C), agents which induce erythroid differentiation. Furthermore, ETS-1 antisense oligonucleotides inhibit hemoglobinization of cells treated with Ara-C or hemin, and K562 and HEL cells infected with retrovirus expressing the c-ETS-1 gene exhibit a significant increase in erythroid character (as indicated by benzidine staining for hemoglobin (Hb) and surface marker analysis), a dramatic increase in responsiveness to hemin or Ara-C, and a decreased rate of proliferation (20-40% of control rates). In contrast, infection with virus expressing ETS-2 or vector sequences only causes no detectable changes in the proliferation or erythroid character of either the HEL or K562 cell lines. These data indicate a role for ETS-1 in erythroid differentiation.

Inhibition of vascular endothelial growth factor-induced endothelial cell migration by ETS1 antisense oligonucleotides.

Vascular endothelial growth factor (VEGF) increased the level of ETS1 mRNA in human umbilical vein endothelial cells (HUVEC) and human lung microvascular endothelial cells (HMVEC-L) over 5-fold. Protein levels were shown to increase concordantly. VEGF was also found to stimulate the invasiveness of endothelial cells as measured by migration through Matrigel- or gelatin-coated membranes. The VEGF-induced invasiveness was inhibited by ETS1 antisense oligonucleotides but not by a sense control. In addition, the ETS1 antisense oligonucleotides reduced the levels of ETS1 and urokinase-type plasminogen activator mRNAs. The antisense oligonucleotides directed against the ETS1 gene thus altered a cellular property of endothelial cells that is correlated with the ability of the cells to migrate through basement membranes. Together, these observations demonstrate a direct role for the ETS1 gene in angiogenesis.

Inversion of a chicken ets-1 proto-oncogene segment in avian leukemia virus E26.

The segment of the avian leukemia virus E26 genome near the termination of the p135gag-myb-ets open reading frame contains an inversion of the chicken ets-1 sequence. The inversion contains at least 41 bp and may be as large as 46 bp. This results in the replacement of 13 amino acids of chicken ets-1, with 16 amino acids derived from reverse complement of the normal ets-1 coding strand or read-through into E26 env sequences. At least 13 of these codons are specified by the inverted ets sequences. This represents the first reported occurrence of inverted oncogene sequences in a natural retrovirus. The inverted ets sequences are immediately followed by sequences homologous to the Rous sarcoma virus Prague B env gene. Since the E26 env sequence is more closely related to subgroup B avian retroviruses than to avian retroviruses from subgroups A, C, D, or E, the progenitor of E26 was a virus belonging to avian retrovirus subgroup B.

Genomic dispersal of the ets gene family during metazoan evolution.

Evolutionary homologs of the ets proto-oncogene have been discovered in the genomes of widely divergent eucaryote species from Drosophila to sea urchin to vertebrates. The prototype mammalian ets-1 and ets-2 genes are divided into three coding domains that differ in their rate of accumulation of sequence divergence. An analysis of sequence divergence of ets gene homologs in various species has produced a phylogenetic history of the ets gene family in the context of metazoan evolutionary radiation. A minimum of five duplication events of ets primordial genes were evident, namely (1) a duplication that separates primitive ets genes (Drosophila precursor of 74E, mouse PU.1 and human ELK1) from the ets-1, ets-2, erg ancestor; (2) and (3) two duplications that established separate ets, erg and elg/GABP-alpha lineages which occurred prior to invertebrate-vertebrate divergence; (4) divergence of ets-1 and ets-2 gene family also associated with vertebrate-invertebrate divergence; (5) duplication of ets-1 and ets-2 in Xenopus laevis to produce two ets-1 genes and two ets-2 genes during genomic tetraploidation in the recent ancestry of this species.

The sea urchin erg homolog defines a highly conserved erg-specific domain.

A genomic clone, isolated from a phage library prepared from the DNA of the sea urchin Lytechinus variegatus, was shown by sequence analysis to be a homolog of the ets family genes, ERG and Fli-1. It contains an open reading frame of which the coding region begins at a consensus 3' splice site and extends for 173 amino acid residues. The first 84 amino acids are homologous with all members of the ets gene family, while the remainder of the sequence is only homologous with the human ERG and murine Fli-1 genes. This latter region, designated R, represents a highly conserved erg-specific domain.

Requirement of ets-2 expression for Xenopus oocyte maturation.

A molecular clone of the Xenopus laevis ets-2 gene was isolated from an oocyte complementary DNA library. The amount of messenger RNA (mRNA) in each oocyte or embryo was almost constant during oogenesis and was maintained until the blastula stage of embryonic development, indicating that the observed 3.2-kilobase transcript is a maternal message. The only normal adult tissue in which ets-2 mRNA was detected was the ovary. Injection of antisense oligonucleotides homologous to the ets-2 sequence into oocytes led to degradation of the mRNA and blocked hormone-induced germinal vesicle breakdown. The ets-2 product is thus required for the meiotic maturation of Xenopus oocytes.

Heterogeneity of Nef proteins in cells infected with human immunodeficiency virus type 1.

Human T-lymphocytic cell line H9 infected with the HTLV-IIIB isolate of human immunodeficiency virus type 1 (HIV-1) synthesizes two forms of the Nef protein (p25 and p27) that differ both in molecular weight and charge. Different subpopulations of viruses were isolated from the HTLV-IIIB stock which induce expression of only p25 or p27. Cells infected with HIV-1 derived from the HXB3 clone of the HTLV-IIIB isolate made only the p25 species, whereas the 8E5/LAV cell line which harbors a single defective LAV provirus produces only the p27 species. These findings are consistent with the notion that the HTLV-IIIB isolate consists of at least two distinct variants with different nef genes, one specifying p25 and the other encoding p27. After a considerable number of passages in culture, H9 cells chronically infected with the HTLV-IIIB isolate produced high levels of p25 and lower levels of p27. Passages in culture appear to select for a subpopulation of virus variants that specify high levels of p25 Nef expression.

Bacterially produced HIV-2 env polypeptides specific for distinguishing HIV-2 from HIV-1 infections.

Five unique recombinant polypeptides, each encoded by a DNA segment representing a different region of the HIV-2 (NIH-Z strain) env gene, were produced at relatively high levels (greater than or equal to 5%) as cII-fusion products in Escherichia coli. These recombinant polypeptides were characterized serologically by the Western blot assay against a panel of HIV-2 and HIV-1 antibody-positive sera, and with normal human sera (HIV-1 and HIV-2 antibody negative). Only those polypeptides that are encoded by a segment of the env gene from the N-terminal region of the transmembrane protein gp35 (amino acids 537 to 707) were immunoreactive. Three polypeptides (921, 996, and 997), each encoding this immunoreactive region of the HIV-2 (NIH-Z) gp35, reacted strongly and specifically with antibodies in sera from HIV-2-positive individuals, but not with antibodies in sera from HIV-1-positive or HIV-uninfected individuals. These results show that the N-terminal region of the HIV-2 gp35 contains a highly antigenic determinant which is strongly immunogenic in HIV-2-infected individuals. The gp35-encoded recombinant env polypeptides can potentially be used in diagnostic assays to specifically differentiate between HIV-2 and HIV-1 infections.

Immunologic detection of protein antigens after phenol-chloroform denaturation.

The study of gene expression in cells and tissues often begins with phenol-chloroform extraction of the biologic material of interest for the isolation of intact mRNA. In most cases, the proteins denatured by phenol-chloroform are discarded. However, we found that the proteins recovered from phenol-chloroform extractions maintain their antigenicity. Therefore a method was developed for recovering the proteins from phenol-chloroform-denatured extracts that could be saved in lyophilized form until immunologic analysis. In this way, the RNA and the protein analysis can utilize exactly the same sample, and the biologic material can be saved. This is important because often these materials are available only in limited quantities. The method has been used to examine the sea urchin ets-related antigen and sea urchin ets-2 mRNA.

Mammalian ets-1 and ets-2 genes encode highly conserved proteins.

Cellular ets sequences homologous to v-ets of the avian leukemia virus E26 are highly conserved. In mammals the ets sequences are dispersed on two separate chromosomal loci, called ets-1 and ets-2. To determine the structure of these two genes and identify the open reading frames that code for the putative proteins, we have sequenced human ets-1 cDNAs and ets-2 cDNA clones obtained from both human and mouse. The human ETS1 gene is capable of encoding a protein of 441 amino acids. This protein is greater than 95% identical to the chicken c-ets-1 gene product. Thus, the human ETS1 gene is homologous to the chicken c-ets-1 gene, the protooncogene that the E26 virus transduced. Human and mouse ets-2 cDNA clones are closely related and contain open reading frames capable of encoding proteins of 469 and 468 residues, respectively. Direct comparison of these data with previously published findings indicates that ets is a family of genes whose members share distinct domains.