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PubMed1981

Clonal analysis of early and late stages of erythroleukemia induced by molecular clones of integrated spleen focus-forming virus.

The integrated proviral DNA of the polycythemia-inducing isolate of Friend spleen focus-forming virus (SFFVp) has been identified in rat cell clones nonproductively infected with this replication-defective erythroleukemia virus and cloned in phage lambda vectors. These lambda SFFVp recombinants, lambda SFFVp502 and lambda SFFVp542, contain endonuclease EcoRI inserts of size 7.4 and 8.2 kilobases, respectively, and include full copies of the SFFVp genome, along with host flanking sequences. Infectivity of the cloned SFFVp genomes was tested by a two-step DNA transfer procedure involving transfection of the cloned DNA into 3T3 mouse fibroblasts or cotransfer of the cloned DNA into thymidine kinase-deficient 3T3 cells together with the cloned thymidine kinase gene of herpes simplex virus, followed by rescue of the transferred DNA by superinfection with a helper virus. Inoculation of the rescued virus into adult mice resulted in the appearance of spleen foci, rapid splenomegaly, and polycythemia. Early after infection, spleen cell populations contained large numbers of cells capable of forming small erythroid colonies in vitro (CFU-E) in the absence of erythropoietin. Late after infection, these mice contained cells capable of forming macroscopic colonies (CFU-FV) in vitro. These data indicate that molecular clones of SFFVp, in conjunction with a helper virus, induce the appearance of hemopoietic colony-forming cells characteristic of both the early and late stages of Friend leukemia.

Animals↗
PubMed1992

Derivation of a biologically contained replication system for human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) proviral mutants that lack viral regulatory genes are unable to replicate unless rescued by complementation in trans. Structurally intact virus can be produced by infecting recombinant cell lines expressing the deficient genes. A HIV-1 mutant functionally defective in tat and rev (vIIIB delta Tat/Rev), which replicates only in a recombinant T-cell line expressing tat and rev (CEMTART), is described in this report. Infection of the CEMTART cell line with vIIIB delta Tat/Rev permits the complete HIV-1 life cycle, including cytopathology, decreased expression of CD4, and production of viral structural proteins, to be biologically contained. Culture supernatants from infected CEMTART contain virus that is able to replicate only in uninfected CEMTART. No reversion of vIIIB delta Tat/Rev to wild-type HIV-1 was observed as measured either by sequencing proviral vIIIB delta Tat/Rev or by detecting the ability of vIIIB delta Tat/Rev to replicate in CEM or activated CD4-bearing T lymphocytes. Defective HIV-1 mutants produced by trans complementation of essential genes permit infection and analysis of defined genotypes on cellular function and phenotype. Authentic HIV-1 structural proteins and infected cells can be prepared in mass, and agents that interfere with the HIV-1 life cycle can be studied on a large scale with minimum risk of exposing workers to virulent HIV-1.

3T3 Cells↗
PubMed1994

CD40-deficient mice generated by recombination-activating gene-2-deficient blastocyst complementation.

To study the role of B-cell antigen CD40 in immune responses, mouse embryonic stem (ES) cells in which both copies of the gene encoding CD40 had been disrupted by homologous recombination were injected in RAG-2 (recombination-activating gene-2)-deficient blastocysts to generate chimeras in which all mature lymphocytes are derived from the CD40-deficient ES cells. T- and B-cell number and phenotype were normal in the CD40-/- chimeras. However, B cells failed to proliferate and undergo isotype switching in vitro in response to soluble CD40 ligand (sCD40L) with interleukin 4 (IL-4) but responded normally to lipopolysaccharide (LPS) with IL-4. CD40-/- chimeras completely failed to mount an antigen-specific antibody response or to develop germinal centers following immunization with the T cell-dependent (TD) antigen keyhole limpet hemocyanin. In contrast, CD40-/- mutant mice responded normally to the T cell-independent (TI) antigens, 2,4,6-trinitrophenyl (TNP)-LPS and TNP-Ficoll. The most noticeable alteration in the serum immunoglobulin levels of young (6-8 weeks old) CD40-/- animals was absence of IgE and severe decrease of IgG1 and IgG2a. These results confirm the essential role of CD40- CD40L interactions in the antibody response to TD antigens and in isotype switching.

Animals↗
PubMed1995

A genetic screen identifies cellular factors involved in retroviral -1 frameshifting.

To identify cellular factors that function in -1 ribosomal frameshifting, we have developed assays in the yeast Saccharomyces cerevisiae to screen for host mutants in which frameshifting is specifically affected. Expression vectors have been constructed in which the mouse mammary tumor virus gag-pro frameshift region is placed upstream of the lacZ gene or the CUP1 gene so that the reporters are in the -1 frame relative to the initiation codon. These vectors have been used to demonstrate that -1 frameshifting is recapitulated in yeast in response to retroviral mRNA signals. Using these reporters, we have isolated spontaneous host mutants in two complementation groups, ifs1 and ifs2, in which frameshifting is increased 2-fold. These mutants are also hypersensitive to antibiotics that target the 40S ribosomal subunit. We have cloned the IFS1 gene and shown that it encodes a previously undescribed protein of 1091 aa with clusters of acidic residues in the carboxyl-terminal region. Haploid cells lacking 82% of the IFS1 open reading frame are viable and phenotypically identical to ifs1-1 mutants. This approach could help identify potential targets for antiretroviral agents.

Adaptor Proteins, Signal Transducing↗
PubMed1996

A chromosomal locus required for copper resistance, competitive fitness, and cytochrome c biogenesis in Pseudomonas fluorescens.

A chromosomal locus required for copper resistance and competitive fitness was cloned from a strain of Pseudomonas fluorescens isolated from copper-contaminated agricultural soil. Sequence analysis of this locus revealed six open reading frames with homology to genes involved in cytochrome c biogenesis in other bacteria, helC, cycJ, cycK, tipB, cycL, and cycH, with the closest similarity being to the aeg-46.5(yej) region of the Escherichia coli chromosome. The proposed functions of these genes in other bacteria include the binding, transport, and coupling of heme to apocytochrome c in the periplasm of these Gram-negative bacteria. Putative heme-binding motifs were present in the predicted products of cycK and cycL, and TipB contained a putative disulfide oxidoreductase active site proposed to maintain the heme-binding site of the apocytochrome in a reduced state for ligation of heme. Tn3-gus mutagenesis showed that expression of the genes was constitutive but enhanced by copper, and confirmed that the genes function both in copper resistance and production of active cytochrome c. However, two mutants in cycH were copper-sensitive and oxidase-positive, suggesting that the functions of these genes, rather than cytochrome c oxidase itself, were required for resistance to copper.

Amino Acid Sequence↗
PubMed1996

Gene-targeted deletion and replacement mutations of the T-cell receptor beta-chain enhancer: the role of enhancer elements in controlling V(D)J recombination accessibility.

To assess the role of transcriptional enhancers in regulating accessibility of the T-cell receptor beta-chain (TCRbeta) locus, we generated embryonic stem cell lines in which a single allelic copy of the endogenous TCRbeta enhancer (Ebeta) was either deleted or replaced with the immunoglobulin heavy-chain intronic enhancer. We assayed the effects of these mutations on activation of the TCRbeta locus in normal T- and B-lineage cells by RAG-2 (recombination-activating gene 2)-deficient blastocyst complementation. We found that Ebeta is required for rearrangement and germ-line transcription of the TCRbeta locus in T-lineage cells. In the absence of Ebeta, the heavy-chain intronic enhancer partially supported joining region beta-chain rearrangement in T- but not in B-lineage cells. However, ability of the heavy-chain intronic enhancer to induce rearrangements was blocked by linkage to an expressed neomycin-resistance gene (neo(r)). These results demonstrate a critical role for Ebeta in promoting accessibility of the TCRbeta locus and suggest that additional negative elements may cooperate to further modulate this process.

Alleles↗
PubMed1996

Single-copy transgenic mice with chosen-site integration.

We describe a general way of introducing transgenes into the mouse germ line for comparing different sequences without the complications of variation in copy number and insertion site. The method uses homologous recombination in embryonic stem (ES) cells to generate mice having a single copy of a transgene integrated into a chosen location in the genome. To test the method, a single copy murine bcl-2 cDNA driven by either a chicken beta-actin promoter or a human beta-actin promoter has been inserted immediately 5' to the X-linked hypoxanthine phosphoribosyltransferase locus by a directly selectable homologous recombination event. The level of expression of the targeted bcl-2 transgene in ES cells is identical in independently isolated homologous recombinants having the same promoter yet varies between the different promoters. In contrast, the expression of bcl-2 transgenes having the same (chicken beta-actin) promoter varies drastically when they are independently integrated at random insertion sites. Both promoters direct broad expression of the single-copy transgene in mice derived from the respective targeted ES cells. In vitro and in vivo, the human beta-actin promoter consistently directed a higher level of transgene expression than the chicken beta-actin promoter.

Animals↗
PubMed1996

Signal transduction through homologs of the Ste20p and Ste7p protein kinases can trigger hyphal formation in the pathogenic fungus Candida albicans.

The CST20 gene of Candida albicans was cloned by functional complementation of a deletion of the STE20 gene in Saccharomyces cerevisiae. CST20 encodes a homolog of the Ste20p/p65PAK family of protein kinases. Colonies of C. albicans cells deleted for CST20 revealed defects in the lateral formation of mycelia on synthetic solid "Spider" media. However, hyphal development was not impaired in some other media. A similar phenotype was caused by deletion of HST7, encoding a functional homolog of the S. cerevisiae Ste7p protein kinase. Overexpression of HST7 partially complemented the deletion of CST20. Cells deleted for CST20 were less virulent in a mouse model for systemic candidiasis. Our results suggest that more than one signaling pathway can trigger hyphal development in C. albicans, one of which has a protein kinase cascade that is analogous to the mating response pathway in S. cerevisiae and might have become adapted to the control of mycelial formation in asexual C. albicans.

Amino Acid Sequence↗
PubMed1995

Substrate specificity and expression profile of amino acid transporters (AAPs) in Arabidopsis.

Three amino acid transporter genes (AAP3-5) were isolated from Arabidopsis by complementation of a yeast mutant defective in histidine uptake. Transport is driven against a concentration gradient and sensitive to protonophores. Analysis of the substrate specificity demonstrates that the carriers have a broad substrate specificity covering the major transport forms of reduced nitrogen, i.e. glutamine and glutamate. The transporters have similar affinities for glutamate, glutamine, and alanine but differ with respect to valine, phenylalanine, histidine, arginine, and lysine. AAP3 and AAP5 efficiently transport arginine and lysine and are involved in basic amino acid transport. The predicted polypeptides of 53 kDa are highly hydrophobic with 12 putative membrane-spanning regions and show significant homologies to Arabidopsis amino acid transporters AAP1 and AAP2. Each of the genes has a different organ-specific expression in the plant. AAP3 is exclusively expressed in roots and AAP4 mainly in source leaves, stems, and flowers, whereas AAP5 is found in all tissues. The specific distribution in the plant and the different substrate specificities of AAP transporters may indicate that tissues differ both qualitatively and quantitatively regarding import or export of amino acids.

Amino Acid Sequence↗
PubMed1996

Arabidopsis thaliana contains two differentially expressed farnesyl-diphosphate synthase genes.

The enzyme farnesyl-diphosphate synthase (FPS; EC 2.5.1.1/EC 2.5.1.10) catalyzes the synthesis of farnesyl diphosphate (FPP) from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This reaction is considered to be a rate-limiting step in isoprenoid biosynthesis. Southern blot analysis indicates that Arabidopsis thaliana contains at least 2 genes (FPS1 and FPS2) encoding FPS. The FPS1 and FPS2 genes have been cloned and characterized. The two genes have a very similar organization with regard to intron positions and exon sizes and share a high level of sequence similarity, not only in the coding region but also in the intronic sequences. Northern blot analysis showed that FPS1 and FPS2 have a different pattern of expression. FPS1 mRNA accumulates preferentially in roots and inflorescences, whereas FPS2 mRNA is predominantly expressed in inflorescences. The cDNA corresponding to the FPS1 gene was isolated by functional complementation of a mutant yeast strain defective in FPS activity (Delourme, D., Lacroute, F., and Karst, F. (1994) Plant Mol. Biol. 26, 1867-1873). By using a reverse transcription-polymerase chain reaction strategy we have cloned the cDNA corresponding to the FPS2 gene. Analysis of the FPS2 cDNA sequence revealed an open reading frame encoding a protein of 342 amino acid residues with a predicted molecular mass of 39,825 Da. FPS1 and FPS2 isoforms share an overall amino acid identity of 90.6%. Arabidopsis FPS2 was able to rescue the lethal phenotype of an ERG20-disrupted yeast strain. We demonstrate that FPS2 catalyzes the two successive condensations of IPP with both DMAPP and geranyl diphosphate leading to FPP. The significance of the occurrence of different FPS isoforms in plants is discussed in the context of the complex organization of the plant isoprenoid pathway.

Alkyl and Aryl Transferases↗
PubMed1998

An acyl-CoA synthase (acoas) gene adjacent to the mycocerosic acid synthase (mas) locus is necessary for mycocerosyl lipid synthesis in Mycobacterium tuberculosis var. bovis BCG.

An open reading frame, ORF3, first identified adjacent to the mycocerosic acid synthase gene in Mycobacterium bovis BCG encodes a protein with acyl-CoA synthase (ACoAS) activity. Genes homologous to acoas are found adjacent to other multifunctional polyketide synthase genes in the mycobacterial genome. To test whether these gene products are necessary to esterify the fatty acids generated by the adjacent polyketide synthase gene products, the acoas gene was disrupted in M. bovis BCG using a suicide vector containing the acoas gene with an internal deletion and the hygromycin-resistant gene as selection marker. Allelic exchange at the acoas locus was confirmed by Southern hybridization and polymerase chain reaction amplification of both flanking regions expected from homologous recombination. Immunoblot analysis indicated that the 65-kDa ACoAS protein product was absent in the mutant. Chromatographic analysis of lipids derived from [1-14C]propionate showed that the mutant did not produce mycocerosyl lipids, although it produced normal levels of mycocerosic acid synthase. These results suggest that ACoAS is involved in the synthesis of mycocerosyl lipids of the mycobacterial cell wall.

Acyltransferases↗
PubMed1996

Distinct regions control transcriptional activation of the alpha1(VI) collagen promoter in different tissues of transgenic mice.

To identify regions involved in tissue specific regulation of transcription of the alpha1(VI) collagen chain, transgenic mice were generated carrying various portions of the gene's 5'-flanking sequence fused to the E. coli beta-galactosidase gene. Analysis of the transgene expression pattern by X-gal staining of embryos revealed that: (a) The proximal 0.6 kb of promoter sequence activated transcription in mesenchymal cells at sites of insertion of superficial muscular aponeurosis into the skin; tendons were also faintly positive. (b) The region between -4.0 and -5.4 kb from the transcription start site was required for activation of the transgene in nerves. It also drove expression in joints, in intervertebral disks, and in subepidermal and vibrissae mesenchyme. (c) The fragment comprised within -6.2 and -7.5 kb was necessary for high level transcription in skeletal muscle and meninges. Positive cells in muscle were mostly mononuclear and probably included connective tissue elements, although staining of myoblasts was not ruled out. This fragment also activated expression in joints, in intervertebral disks, and in subepidermal and vibrissae mesenchyme. (d) beta-Galactosidase staining in vibrissae induced by the sequences -4.0 to -5.4 and -6.2 to -7.5 was not coincident: with the latter sequence labeled nuclei were found mainly in the ventral and posterior quadrant, and, histologically, in the outer layers of mesenchyme surrounding and between the follicles, whereas with the former the remaining quadrants were positive and expressing cells were mostly in the inner layers of the dermal sheath. (e) Other tissues, notably lung, adrenal gland, digestive tract, which produce high amounts of collagen type VI, did not stain for beta-galactosidase. (f) Central nervous system and retina, in which the endogenous gene is inactive, expressed the lacZ transgene in most lines. The data suggest that transcription of alpha1(VI) in different tissues is regulated by distinct sequence elements in a modular arrangement, a mechanism which confers high flexibility in the temporal and spatial pattern of expression during development.

Animals↗
PubMed2000

Genomewide scan for prostate cancer-aggressiveness loci.

The aggressiveness of prostate cancer (PCa) varies widely: some tumors progress to invasive, potentially life-threatening disease, whereas others stay latent for the remainder of an individual's lifetime. The mechanisms resulting in this variability are not yet understood, but they are likely to involve both genetic and environmental influences. To investigate genetic factors, we conducted a genomewide linkage analysis of 513 brothers with PCa, using the Gleason score, which reflects tumor histology, as a quantitative measure of PCa aggressiveness. To our knowledge, this is the first time that a measure of PCa aggressiveness has been directly investigated as a quantitative trait in a genomewide scan. We employed a generalized multipoint Haseman-Elston linkage-analysis approach that regresses the mean-corrected cross product between the brothers' Gleason scores on the estimated proportion of alleles shared by brothers identical by descent at each marker location. Our results suggest that candidate regions on chromosomes 5q, 7q, and 19q give evidence for linkage to PCa-aggressiveness genes. In particular, the strongest signals detected in these regions were at the following markers (with corresponding P values): for chromosome 5q31-33, between markers D5S1480 and D5S820 (P=.0002); for chromosome 7q32, between markers D7S3061 and D7S1804 (P=.0007); and, for chromosome 19q12, at D19S433 (P=.0004). This indicates that one or more of these candidate regions may contain genes that influence the progression of PCa from latent to invasive disease. Identification of such genes would be extremely valuable for elucidation of the mechanism underlying PCa progression and for determination of treatment in men in whom this disease has been diagnosed.

Adult↗
PubMed2002

A genomewide linkage screen for relative hand skill in sibling pairs.

Genomewide quantitative-trait locus (QTL) linkage analysis was performed using a continuous measure of relative hand skill (PegQ) in a sample of 195 reading-disabled sibling pairs from the United Kingdom. This was the first genomewide screen for any measure related to handedness. The mean PegQ in the sample was equivalent to that of normative data, and PegQ was not correlated with tests of reading ability (correlations between minus sign0.13 and 0.05). Relative hand skill could therefore be considered normal within the sample. A QTL on chromosome 2p11.2-12 yielded strong evidence for linkage to PegQ (empirical P=.00007), and another suggestive QTL on 17p11-q23 was also identified (empirical P=.002). The 2p11.2-12 locus was further analyzed in an independent sample of 143 reading-disabled sibling pairs, and this analysis yielded an empirical P=.13. Relative hand skill therefore is probably a complex multifactorial phenotype with a heterogeneous background, but nevertheless is amenable to QTL-based gene-mapping approaches.

Chromosome Mapping↗
PubMed2003

A genomewide screen for generalized vitiligo: confirmation of AIS1 on chromosome 1p31 and evidence for additional susceptibility loci.

Generalized vitiligo is a common autoimmune disorder characterized by the development of white patches of skin and overlying hair due to loss of pigment-forming melanocytes from the involved areas. Family clustering of cases is not uncommon, in a pattern suggestive of multifactorial, polygenic inheritance, and there is strong association between vitiligo and other autoimmune diseases. To map genetic loci that confer susceptibility to generalized vitiligo and perhaps other autoimmune diseases, we performed a genomewide linkage scan in 71 white multiplex families with vitiligo from North America and the United Kingdom. Linkage was assessed by multipoint nonparametric linkage analyses. One linkage signal, AIS1, located at 1p31, met genomewide criteria for highly significant linkage (nonparametric LOD 5.56; P=.000000282), establishing its importance as a major vitiligo susceptibility locus. An additional seven signals, on chromosomes 1, 7, 8, 11, 19, and 22, met genomewide criteria for "suggestive linkage," and will thus be of particular importance for follow-up studies.

Chromosomes, Human, Pair 1↗
PubMed2003

Predisposition locus for major depression at chromosome 12q22-12q23.2.

Major depression disorder is a common psychiatric disease with a major economic impact on society. In many cases, no effective treatment is available. The etiology of major depression is complex, but it is clear that the disease is, to a large extent, determined genetically, especially among individuals with a familial history of major depression, presumably through the involvement of multiple predisposition genes in addition to an environmental component. As a first step toward identification of chromosomal loci contributing to genetic predisposition to major depression, we have conducted a genomewide scan by using 628 microsatellite markers on 1,890 individuals from 110 Utah pedigrees with a strong family history of major depression. We identified significant linkage to major depression in males at marker D12S1300 (multipoint heterogeneity LOD score 4.6; P=.00003 after adjustment for multiple testing). With additional markers, the linkage evidence became highly significant, with the multipoint heterogeneity LOD score at marker D12S1706 increasing to 6.1 (P=.0000007 after adjustment for multiple testing). This study confirms the presence of one or more genes involved in psychiatric diseases on the q arm of chromosome 12 and provides strong evidence for the existence of a sex-specific predisposition gene to major depression at 12q22-q23.2.

Chromosome Mapping↗