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At least 667 records · Page 37Linked to original sources
PubMed1989

Heterogeneity within an HSV-1 wild-type strain and its importance in pathogenesis.

A herpes simplex virus-type 1 low passage, clinical eye isolate, E-43 at P2, was compared with its variant progeny, SLi-43 at P8, in terms of ocular disease, cytopathic effects, and genomic variation. In New Zealand White (NZW) rabbits, E-43 produced mild epithelial defects and punctate lesions with full recovery by Day 10 postinfection (pi). SLi-43 caused dendritic lesions, progressing to geographic ulceration and death from herpes simplex virus encephalitis in 10 days postinfection. In RK, Hep-2, and Vero cells, E-43 displayed the syn+ phenotype (aggregation and cell rounding); SLi-43 showed the syn phenotype (syncytium formation). DNA digestion profiles of E-43, SLi-43, and isolates from the brains of infected animals showed that the genomic differences map within the terminal repeat of the unique long segment and the internal joint region, specifically in bands B, E, N, and S (Bam HI) and bands M and N (Hind III). Analysis of the DNA of virus recovered from the brain stem of SLi-43-infected, encephalitic rabbits demonstrated that an in vivo selection for neurotropic virions had taken place. Plaque purification of 20 clones from the original E-43 strain showed that one of 20 was the syn phenotype, indicating that the SLi-43 variant was present in the original E-43 isolate and did not develop de novo by rapid mutation. The parent-progeny relationship between E-43 and SLi-43 forms an ideal model in which to compare differences in pathogenicity at the genomic level, and underscores the importance of heterogeneity within a single herpes simplex virus-type 1 wild-type population in terms of variations in ocular disease.

Animals↗
PubMed1988

Native genomic blotting: a novel approach to mapping DNase I hypersensitive sites and protein-DNA interactions at high resolution.

We have developed a new high resolution method for screening 400-600 base pairs of DNA in chromatin for DNase I hypersensitive sites and protein-DNA interactions. By separating the DNA isolated from nuclease-digested nuclei in small, native polyacrylamide gels prior to electroblotting onto nylon membranes, we increased the resolution by greater than 3-fold as compared with the traditional approach whereby the nuclease-digested DNA is fractionated electrophoretically in agarose gels (11). In addition, our native genomic blotting method has the advantage of combining the ability of the traditional agarose approach to detect DNase I hypersensitive sites, with the genomic sequencing method (2), where individual protein-DNA contacts can be observed. Native genomic blotting therefore permits for the first time the display of DNase I hypersensitive sites and protein-DNA interactions at high resolution on the same autoradiograph. This method allows us to investigate a new level of chromatin structure and to therefore obtain better insight into levels of gene structure, organization and gene regulation.

Autoradiography↗
PubMed1989

Functional analysis of GC element binding and transcription in the hamster dihydrofolate reductase gene promoter.

Dihydrofolate reductase (DHFR) gene expression is required for cell growth. The DHFR gene promoter contains several GC elements capable of binding the transcription factor Sp1. In this report we have characterized the effect of protein(s) binding to these sequence elements in the Chinese hamster DHFR promoter on transcription. We have constructed a series of deletions containing from 896 to 103 bp 5' to the start of translation. The protein binding domains have been mapped by DNAse I footprint analysis using HeLa nuclear extract, and the function of the protein-binding elements has been assessed by in vitro transcription and transient CAT expression. Maximal transcription in vitro and CAT expression is obtained with a construct containing 3 GC elements extending to position -184. Removal of GC element binding factor(s), by competition with an oligonucleotide containing an Sp1 binding site, completely abolishes transcription in vitro and significantly diminishes CAT expression. Ten-fold higher molar excess of competitor is required to abolish SV40 early transcription, suggesting that the GC element interactions in the DHFR promoter are different from those in the SV40 early region. Co-transfection of a DHFR CAT construct with an expressor of Sp1 dramatically increased CAT expression in Drosophila cells.

Animals↗
PubMed1988

Human ornithine-delta-aminotransferase. cDNA cloning and analysis of the structural gene.

Ornithine-delta-aminotransferase (OAT) is a nuclear-encoded, mitochondrial matrix enzyme which, in rat, is expressed as basal levels in most tissues but is induced in liver by high dietary protein and in kidney by estrogen and thyroxine administration. In man, the hereditary deficiency of OAT results in ornithine accumulation and the blinding disease gyrate atrophy of the choroid and retina. We cloned near full length rat and human liver OAT cDNAs and demonstrated OAT expression in a variety of tissues from each species. We mapped the human OAT structural gene to chromosome 10, cloned 40 kilobase pairs of genomic DNA containing the complete OAT structural gene, and determined its organization. It is 21 kilobase pairs in length, and contains 11 exons. Exon 2 has been absent from all cDNAs studied and was detected by homology to X-linked processed OAT pseudogenes. The 5'-flanking region of the OAT gene has features of housekeeping genes (GC enrichment and three Sp1 binding consensus sequences) and tissue-specific, inducible genes (TATA box-like element and two CCAAT boxes). A 22-base pair region of partial dyad symmetry containing homology to estrogen responsive elements overlaps the OAT transcription site. Another 5' sequence, GTATCCTGCCCTC, is homologous to sequences in the promoter regions of the genes of three urea cycle enzymes.

Amino Acid Sequence↗
PubMed1988

Native genomic blotting: high-resolution mapping of DNase I-hypersensitive sites and protein-DNA interactions.

DNase I-hypersensitive sites are observed in the promoter regions of actively expressed genes, potentially active genes, and genes that were once active. We have developed an approach that greatly increases the resolution for mapping these sites by electrophoresing genomic DNA on native polyacrylamide gels prior to electroblotting and hybridization. This improved method has been used to scan the promoter and coding region of a cell-cycle-dependent human histone H4 gene with an accuracy of +/-5-10 base pairs. Protein-DNA interactions can be seen in the autoradiograph as light areas and DNase I-hypersensitive sites as dark bands. Therefore, this method provides a rapid and relatively simple means to accurately localize protein-DNA interactions as well as DNase I-hypersensitive sites, thus directly displaying DNase I hypersensitivity and protein-DNA complexes on one autoradiograph. It also potentially allows the analysis of small changes in DNase I-hypersensitive sites under various biological conditions. With this technique rather large regions of DNA can be screened to determine areas that should be analyzed by more sophisticated methods, such as genomic sequencing or gel retardation assays.

DNA, Neoplasm↗
PubMed2004

Large-scale association study identifies ICAM gene region as breast and prostate cancer susceptibility locus.

We conducted a large-scale association study to identify genes that influence nonfamilial breast cancer risk using a collection of German cases and matched controls and >25,000 single nucleotide polymorphisms located within 16,000 genes. One of the candidate loci identified was located on chromosome 19p13.2 [odds ratio (OR) = 1.5, P = 0.001]. The effect was substantially stronger in the subset of cases with reported family history of breast cancer (OR = 3.4, P = 0.001). The finding was subsequently replicated in two independent collections (combined OR = 1.4, P < 0.001) and was also associated with predisposition to prostate cancer in an independent sample set of prostate cancer cases and matched controls (OR = 1.4, P = 0.002). High-density single nucleotide polymorphism mapping showed that the extent of association spans 20 kb and includes the intercellular adhesion molecule genes ICAM1, ICAM4, and ICAM5. Although genetic variants in ICAM5 showed the strongest association with disease status, ICAM1 is expressed at highest levels in normal and tumor breast tissue. A variant in ICAM5 was also associated with disease progression and prognosis. Because ICAMs are suitable targets for antibodies and small molecules, these findings may not only provide diagnostic and prognostic markers but also new therapeutic opportunities in breast and prostate cancer.

Adult↗
PubMed1990

Complementation analysis of Agrobacterium tumefaciens Ti plasmid virB genes by use of a vir promoter expression vector: virB9, virB10, and virB11 are essential virulence genes.

The virB gene products of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid have been proposed to mediate T-DNA transport through the bacterial cell wall into plant cells. Previous genetic analysis of the approximately 9.5-kilobase-pair virB operon has been limited to transposon insertion mutagenesis. Due to the polarity of the transposon insertions, only the last gene in the operon, virB11, is known to provide an essential virulence function. We have now begun to assess the contribution of the other virB genes to virulence. First, several previously isolated Tn3-HoHo1 insertions in the 3' end of the virB operon were precisely mapped by nucleotide sequence analysis. Protein extracts from A. tumefaciens strains harboring these insertions on the Ti plasmid were subjected to immunostaining analysis with VirB4-, VirB10-, and VirB11-specific antisera to determine the effect of the insertion on virB gene expression. In this manner, avirulent mutants containing polar insertions in the virB9 and virB10 genes were identified. To carry out a complementation analysis with these virB mutants, expression vectors were constructed that allow cloned genes to be expressed from the virB promoter in A. tumefaciens. These plasmids were used to express combinations of the virB9, virB10, and virB11 genes in trans in the virB insertion mutants, thereby creating strains lacking only one of these three virB gene products. Virulence assays on Kalanchoe daigremontiana demonstrated that in addition to virB11, the virB9 and virB10 genes are required for tumorigenicity.

Bacterial Proteins↗
PubMed1992

Mutational analysis of the ICP4 binding sites in the 5' transcribed noncoding domains of the herpes simplex virus 1 UL 49.5 gamma 2 gene.

A previous report (P. Mavromara-Nazos and B. Roizman, Proc. Natl. Acad. Sci. USA 86:4071-4075, 1989) demonstrated that substitution of sequences of the thymidine kinase (tk) gene, a beta gene, extending from -16 to +51 with sequences extending from -12 to +104 of the gamma 2 UL 49.5 gene in viral recombinant R3820 conferred upon the chimeric gene gamma 2 attributes in the context of the viral genome in a productive infection. The UL49.5 gene sequences extending from -179 to +104 contain four DNA binding sites for the major regulatory protein ICP4. Of these sites, two map between nucleotides +20 and +80 within the sequence which confers gamma 2 regulation upon the chimeric gene. To determine the role of these ICP4 binding sites in conferring the gamma 2 gene attributes, sequences comprising the two ICP4 binding sites were mutagenized and used to reconstruct the R3820 recombinant virus. In addition, a new recombinant virus (R8023) was constructed in which tk sequences extending from -240 to +51 were replaced with wild-type or mutated sequences contained between nucleotides -179 to +104 of the UL 49.5 gene. Vero cells infected with the recombinant viruses in the presence or absence of phosphonoacetate, a specific inhibitor of viral DNA synthesis, were then tested for accumulation of tk RNA by using an RNase protection assay. The results indicate that in the recombinant R3820, a mutation which destroyed one of the two UL49.5 ICP4 DNA binding sites significantly reduced the accumulation of tk RNA at both early and late times after infection. The effect of this mutation was less pronounced in cells infected with the R8023 virus, whose chimeric tk gene contains the two upstream UL49.5 ICP4 binding sites. None of the mutations affected the sensitivity of the chimeric genes to phosphonoacetate. The mutated site appears to be involved in the accumulation of RNA.

Animals↗
PubMed1989

Structure of the three beta-tubulin-encoding genes of the unicellular alga, Polytomella agilis.

The quadriflagellate, unicellular, colorless alga, Polytomella agilis, contains several distinct microtubule arrays. To study the genetic basis of microtubule heterogeneity in P. agilis, we characterized its tubulin(Tub)-encoding genes (tub). The three beta tub genes detected in blots of P. agilis DNA were isolated from a genomic library. The structure and organization of the genes were examined by restriction mapping and nucleotide (nt) sequencing. S1 nuclease protection studies showed that all three genes are expressed. The predicted amino acid (aa) sequences are more than 98% conserved with the Chlamydomonas reinhardtii and Volvox carteri beta-Tubs, underscoring the close phylogenetic relationship of these species. Evolutionary divergence among the P. agilis genes is demonstrated by differences in intron number, nt sequences in noncoding regions, and silent nt substitutions in the coding regions. However, the proteins encoded by the beta 1 and beta 3 tub genes are identical; the beta 2 gene product differs by one conservative aa substitution. These results are in striking contrast to the C-terminal aa diversity reported within beta tub gene families in animal, higher plant and fungal systems. The data support the hypothesis that those tub genes whose products assemble into axonemal microtubules are subject

Amino Acid Sequence↗
PubMed2000

Identification of novel residues involved in nuclear localization of a baculovirus polyhedrin protein.

A baculovirus polyhedrin protein has proven to possess a nuclear localization signal (NLS) sequence and a domain required for supramolecular assembly. Here we investigated five Bombyx mori nucleopolyhedrovirus (BmNPV) mutants that did not produce polyhedra. Two of five mutants were generated during routine baculoviral expression vector screening, and three were isolated by treatment with the mutagen 5-bromo-2'-deoxyuridine (BrdU). Marker rescue mapping and nucleotide sequence analysis showed that mutations in the polyhedrin gene caused the altered phenotype of these mutants. Biochemical fractionation indicated that cells infected with these mutants exhibited polyhedrin protein in both the nucleus and the cytoplasm. Electron microscopic observation revealed that polyhedrin produced by these mutants ocurred in both the nucleus and the cytoplasm, but did not form a crystalline lattice. Despite the incompleteness of polyhedrin nuclear localization, the NLSs of the five mutants were unchanged, although some of the mutations occurred within residues just outside of the domain reported to be required for polyhedron assembly (4). This result suggests that (a) the polyhedrin NLS directs polyhedrin to the nucleus, but the efficiency of this localization is regulated by regions other than the NLS (probably, polyhedrin conformation and its association with the nucleus are also involved), and (b) formation of a crystalline lattice may also be determined by several domains within polyhedrin.

Animals↗
PubMed1991

Control of bacteriophage P2 gene expression: analysis of transcription of the ogr gene.

The bacteriophage P2 ogr gene encodes an 8.3-kDa protein that is a positive effector of P2 late gene transcription. The ogr gene is preceded by a promoter sequence (Pogr) resembling a normal Escherichia coli promoter and is located just downstream of a late transcription unit. We analyzed the kinetics and regulation of ogr gene transcription by using an ogr-specific antisense RNA probe in an S1 mapping assay. During a normal P2 infection, ogr gene transcription starts from Pogr at an intermediate time between the onset of early and late transcription. At late times after infection the ogr gene is cotranscribed with the late FETUD operon; the ogr gene product thus positively regulates its own synthesis from the P2 late promoter PF. Expression of the P2 late genes also requires P2 DNA replication. Complementation experiments and transcriptional analysis show that a nonreplicating P2 phage expresses the ogr gene from Pogr but is unable to transcribe the late genes. A P2 ogr-defective phage makes an increased level of ogr mRNA, consistent with autogenous control from Pogr. Transcription of the ogr gene in the prophage of a P2 heteroimmune lysogen is stimulated after infection with P2, suggesting that Pogr is under indirect immunity control and is activated by a yet-unidentified P2 early gene product during infection.

Coliphages↗
PubMed1988

Fine mapping of an H-2Kk restricted cytotoxic T lymphocyte epitope in SV40 T antigen by using in-frame deletion mutants and a synthetic peptide.

The CTL response to SV40 in C3H/HeJ mice is directed against the tumor (T) Ag and is H-2Kk restricted. CTL specific for both the amino terminus (residues 1-271) and the carboxyl terminus (residues 512-708) of the T Ag molecule have been detected, and we have previously cloned CTL of both specificities. In this paper we show that the panel of 10 CTL clones specific for the C-terminal region includes clones specific for three different epitopes, termed C1, C2, and C3. Epitopes C1 and C2 are conserved in the T Ag of the related papova viruses BK and SA12, and only epitopes C2 and C3 are present on SV40 transformed targets bearing the Kk mutant Kkml. Epitopes C1 and C2 were mapped to residues 563-576 by using in-frame deletion mutants of SV40 T antigen, and all clones specific for these two epitopes can lyse Kk bearing target cells in the presence of a synthetic peptide comprising residues 559-576. Kk and Kkml differ at residue 152, which is located in the Ag-binding pocket. Because epitopes C1 and C2 can be formed by the same antigenic peptide, but epitope C1 is not present on SV40 transformed Kkml cells, epitopes C1 and C2 must differ in the contribution made by residue 152 of the MHC class I molecule. These data show that CTL epitopes on transformed cells can be made up of Ag fragments, and strengthen the idea that this is a general phenomenon for both class I and class II restricted T cell epitopes.

Amino Acid Sequence↗
PubMed1980

Mode of inheritance and evidence for cistron heterogeneity of chloroplast 16S ribosomal RNA genes in Nicotiana.

Oligonucleotide maps (fingerprints) of T1 RNase digests of 125I-labeled 16 S chloroplast rRNA of Nicotiana tabacum and N. gossei revealed the presence of T1 oligonucleotide fragment 100 in the 16 S rRNA of N. gossei while N. tabacum 16 S rRNA had a unique T1 oligonucleotide (fragment 101) as well as some fragment 100. From the positions in the fingerprints and from fingerprints of secondary enzymatic digestion of the fragments, we conclude that fragments 100 and 101 are similar in sequence and size, but fragment 100 probably contains an extra uracil residue. This difference is shown to be maternally inherited, thus confirming the location of 16 S chloroplast rRNA genes on chloroplast DNA and ruling out the possibility of genetically active chloroplast rRNA genes in the nucleus. The presence of both fragments 100 and 101 in N. tabacum may indicate sequence heterogeneity between the two cistrons for 16 S chloroplast rRNA. These results demonstrate the feasibility of determining the inheritance of organelle genes by genetic analysis of their primary transcripts.

Chloroplasts↗
PubMed1990

The complete and symmetric transcription of the main non coding region of rat mitochondrial genome: in vivo mapping of heavy and light transcripts.

The experiments here reported demonstrate that the main non-coding region of rat mitochondrial DNA is symmetrically transcribed. We have identified stable heavy and light transcripts, whose pattern is rather complex, in the D-loop region of rat mitochondrial DNA. Their relative concentrations have been determined. We detected heavy transcripts which encompass the whole D-loop and more abundant heavy RNA species which we interpreted as transcripts terminating downstream of the 3' end of the last coded gene (Thr-tRNA). The processed heavy RNA species contain polyA, suggesting a strict association between cleavage and polyadenylation. The pattern of light transcripts shows a long RNA, which, starting from the light strand promoter, covers the whole segment, and shorter RNA species which seems to be actively processed at the level of the conserved sequence boxes, probably acting as primers. The symmetric transcription of the D-loop containing region of rat mitochondrial DNA, and in particular the presence of stable transcripts complementary to the putative RNA primers, suggest that mechanisms mediated by interaction between complementary transcripts (antisense RNAs) might play a role in the regulation of mitochondrial DNA replication and expression.

Amino Acid Sequence↗
PubMed1991

In vitro immunization of human B lymphocytes. MAPPing of lymphokine specific mRNA and the effect of recombinant factors.

The kinetics of lymphokine-specific DNA transcription during in vitro immunization of human peripheral blood lymphocytes and splenocytes were studied using the polymerase chain reaction. The levels of specific mRNA were shown to be down regulated by cytolytic L-leucyl-leucine methyl ester-sensitive lymphocytes. In in vitro immunizations using L-leucyl-leucine methyl ester-treated human PBL or splenocytes, the lymphokine mRNA expression pattern indicated an active gene transcription during the entire stimulation period, especially for the IL-2 and IL-5 genes. Transcription of IL-6 and TNF beta started on day 4, whereas IFN gamma mRNA reached its maximum level on day 4. In vitro immunizations of cells not treated with L-leucyl-leucine methyl ester revealed a transient transcription of lymphokine DNA that was declining already after day 2. Exogenously added recombinant IL-2, IL-4, and IL-6 all exhibited a positive immunoregulatory effect on Ig secretion, whereas IL-5 was not found to have any effect on immunoglobulin secretion during the in vitro culture. These results present the first information useful for designing in vitro immunization systems based on recombinant lymphokines and antisense DNA for gene regulation.

B-Lymphocytes↗
PubMed1990

Tissue- and species-specific promoter elements of rat gamma-crystallin genes.

The 5' flanking regions of the six rat gamma-crystallin genes (gamma A-gamma F) are all capable of conferring lens-specific expression to the bacterial chloramphenicol acetyl transferase (CAT) reporter gene in either transdifferentiating chicken neural retina cells or mouse lens epithelial cells. Deletion mapping of the most active gamma-crystallin promoter region, the gamma D region, showed that at least three elements are required for maximal expression in mouse lens epithelial cells: element(s) located between -200 and -106, a conserved CG rich region around position -75, and a CG stretch around -15. The region between -200 and -106 was dispensable in transdifferentiating chicken neural retina cells, which instead required the region between -106 and -78. The maximal activity of the gamma E and gamma F promoters was also dependent upon the integrity of the conserved CG region located around -75. A synthetic oligonucleotide containing this sequence was capable of lens-specific enhancement of the activity of the tk promoter in transdifferentiating chicken neural retina cells but not in mouse lens epithelial cells. Our results further show that this region may contain a silencer element, active in non-lens tissues, as well.

Animals↗
PubMed1990

Mouse insulin-responsive glucose transporter gene: characterization of the gene and trans-activation by the CCAAT/enhancer binding protein.

Adipose tissue and skeletal and heart muscle, which exhibit insulin-stimulated glucose uptake, express a specific, insulin-responsive glucose transporter. Previously, a cDNA (GT2) encoding this protein was isolated from a mouse 3T3-L1 adipocyte library and was sequenced. Here we report the isolation and characterization of the corresponding mouse gene designated GLUT4. The GLUT4 gene spans 7 kilobases and consists of 11 exons and 10 introns. The start site of transcription was mapped 180 nucleotides upstream of the initial methionine codon. The GLUT4 promoter contains four potential binding sites for the nuclear transcription factor Sp1 as well as a CCAAT box. DNase I footprinting of the GLUT4 promoter with nuclear extracts from undifferentiated and differentiated 3T3-L1 cells revealed that a differentiation-specific nuclear factor binds in the region at position -258 relative to the start site of transcription. Purified CCAAT/enhancer binding protein (C/EBP) was found to bind at the same position. Transient cotransfection into 3T3-L1 preadipocytes of a GLUT4 promoter-chloramphenicol acetyltransferase gene construct that contains the C/EBP binding site, together with a C/EBP expression vector, revealed that C/EBP trans-activates the GLUT4 promoter. We suggest that C/EBP plays an important role in tissue-specific, as well as metabolic, regulation of the insulin-responsive glucose transporter gene.

Adipose Tissue↗
PubMed1989

Sequence and characteristics of the Bifidobacterium longum gene encoding L-lactate dehydrogenase and the primary structure of the enzyme: a new feature of the allosteric site.

The gene ldh, encoding L-lactate dehydrogenase (LDH; EC 1.1.1.27) of Bifidobacterium longum aM101-2, was cloned in Escherichia coli using an oligodeoxyribonucleotide hybridization probe. The amino acid (aa) sequence, deduced from the sequence of the cloned DNA, was consistent with the results of protein chemical analysis of B. longum LDH. The transcription start points (tsp) in B. longum were identified by S1 nuclease mapping. A sequence, GTAGCAA-(14 bp)-TTATAGA, which is located a few bp upstream from the tsp, was assigned as the promoter of this ldh gene. In the 3'-noncoding region, there were two structures that strongly resembled the Rho-independent transcriptional termination signal of E. coli. Therefore, the B. longum ldh gene might form a monocistronic unit. The deduced primary structure of B. longum LDH had 40% identity with LDHs from Thermus caldophilus, Bacillus stearothermophilus, Lactobacillus casei and dogfish muscle. Most bacterial LDHs are allosterically regulated by fructose 1,6-bisphosphate (FBP), while the vertebrate LDHs are not. The anion-binding site of vertebrate LDHs has been thought to correspond to the FBP-binding site of bacterial LDHs. Although the B. longum LDH was regulated by FBP, the charge properties of aa residues in the putative FBP-binding site of the LDH were closer to those of the vertebrate LDHs than to those of bacterial LDHs.

Allosteric Site↗